Luminescent compounds

ABSTRACT

Dyes and photoluminescent compounds based on polymethine dyes that contain at least one alkyl-phosphonate or substituted alkyl-phosphonate group, including the synthetic precursors, methods of synthesis, and applications thereof. Certain embodiments include heterocyclic ring systems and polymethine linkages selected such that the resulting polymethine dye is a cyanine dye, a merocyanine dye or a styryl dye.

CROSS-REFERENCE TO RELATED APPLICATIONS

This application is a continuation of and claims priority to U.S. patentapplication Ser. No. 13/144,890, filed on Sep. 20, 2011 which is anational stage filing under 35 U.S.C. 371 of International ApplicationNo. PCT/US2010/021282, filed Jan. 15, 2010, which claims the benefit ofU.S. Provisional Application No. 61/145,045, filed Jan. 15, 2009. Thecomplete disclosures of the above-identified applications areincorporated herein by reference for all purposes.

CROSS-REFERENCES TO RELATED MATERIALS

This application incorporates by reference in their entirety for allpurposes all patents, patent applications (published, pending, and/orabandoned), and other patent and nonpatent references cited anywhere inthis application. The cross-referenced materials include but are notlimited to the following publications: Richard P. Haugland, HANDBOOK OFFLUORESCENT PROBES AND RESEARCH CHEMICALS (6^(th) ed. 1996); JOSEPH R.LAKOWICZ, PRINCIPLES OF FLUORESCENCE SPECTROSCOPY (2^(nd) Ed. 1999);RICHARD J. LEWIS, SR., HAWLEY'S CONDENSED CHEMICAL DICTIONARY (12^(th)ed. 1993).

TECHNICAL FIELD

The invention relates to compounds based on cyanines, among others. Moreparticularly, the invention relates to compounds based on cyaninescontaining alkyl-phosphonate and substituted alkyl-phosphonate residuesincluding biologically active phosphate esters that are useful as dyesand luminescent reporters for assays and cell-based applications.

BACKGROUND

Colorimetric and/or luminescent compounds may offer researchers theopportunity to use color and light to analyze samples, investigatereactions, and perform assays, either qualitatively or quantitatively.Generally, brighter, more photostable reporters may permit faster, moresensitive, and more selective methods to be utilized in such research.

While a colorimetric compound absorbs light, and may be detected by thatabsorbance, a luminescent compound, or luminophore, is a compound thatemits light. A luminescence method, in turn, is a method that involvesdetecting light emitted by a luminophore, and using properties of thatlight to understand properties of the luminophore and its environment.Luminescence methods may be based on chemiluminescence and/orphotoluminescence, among others, and may be used in spectroscopy,microscopy, immunoassays, and hybridization assays, among others.

Photoluminescence is a particular type of luminescence that involves theabsorption and subsequent re-emission of light. In photoluminescence, aluminophore is excited from a low-energy ground state into ahigher-energy excited state by the absorption of a photon of light. Theenergy associated with this transition is subsequently lost through oneor more of several mechanisms, including production of a photon throughfluorescence or phosphorescence.

Photoluminescence may be characterized by a number of parameters,including extinction coefficient, excitation and emission spectrum,Stokes' shift, luminescence lifetime, and quantum yield. An extinctioncoefficient is a wavelength-dependent measure of the absorbing power ofa luminophore. An excitation spectrum is the dependence of emissionintensity upon the excitation wavelength, measured at a single constantemission wavelength. An emission spectrum is the wavelength distributionof the emission, measured after excitation with a single constantexcitation wavelength. A Stokes' shift is the difference in wavelengthsbetween the maximum of the emission spectrum and the maximum of theabsorption spectrum. The luminescence lifetime is the average time thata luminophore spends in the excited state prior to returning to theground state and emission of a photon. A quantum yield is the ratio ofthe number of photons emitted to the number of photons absorbed by aluminophore.

Luminescence methods may be influenced by extinction coefficient,excitation and emission spectra, Stokes' shift, and quantum yield, amongothers, and may involve characterizing fluorescence intensity,fluorescence polarization (FP), fluorescence resonance energy transfer(FRET), fluorescence lifetime (FLT), total internal reflectionfluorescence (TIRF), fluorescence correlation spectroscopy (FCS),fluorescence recovery after photobleaching (FRAP), and theirphosphorescence analogs, among others.

Luminescence methods have several significant potential strengths.First, luminescence methods may be very sensitive, because moderndetectors, such as photomultiplier tubes (PMTs) and charge-coupleddevices (CCDs), can detect very low levels of light. Second,luminescence methods may be very selective, because the luminescencesignal may come almost exclusively from the luminophore.

Despite these potential strengths, luminescence methods may suffer froma number of shortcomings, at least some of which relate to theluminophore. For example, the luminophore may have an extinctioncoefficient and/or quantum yield that is too low to permit detection ofan adequate amount of light. The luminophore also may have a Stokes'shift that is too small to permit effective detection of emission lightwithout significant detection of excitation light. The luminophore alsomay have an excitation spectrum that does not permit it to be excited bywavelength-limited light sources, such as common lasers and arc lamps.The luminophore also may be unstable, so that it is readily bleached andrendered nonluminescent. The luminescent compound may not be passivelyable to pass the plasma membrane in cells due to the presence of one ormore ionic charges. The luminophore also may have an excitation and/oremission spectrum that overlaps with the well-known autoluminescence ofbiological and other samples; such autoluminescence is particularlysignificant at wavelengths below about 600 nm. The luminophore also maybe expensive, especially if it is difficult to manufacture.

SUMMARY

The invention relates generally to dyes and photoluminescent compoundsbased on polymethine dyes that contain at least one alkyl-phosphonate orsubstituted alkyl-phosphonate group, including the synthetic precursors,methods of synthesis, and applications thereof.

ABBREVIATIONS

The following abbreviations, among others, may be used in thisapplication:

Abbreviation Definition BSA bovine serum albumin Bu butyl DCCdicyclohexylcarbodiimide DMF dimethylformamide D/P dye-to-protein ratioEt ethyl g grams h hours HSA human serum albumin IgG Immunoglobulin G Lliters m milli (10⁻³) M molar Me methyl mol moles M.P. melting point nmnanometer (10⁻⁹ meter) NHS N-hydroxysuccinimide NIR near infrared PBSphosphate-buffered saline Prop propyl TMS tetramethylsilane TSTUN,N,N′,N′-tetramethyl(succinimido)uronium tetra- fluoroborate μ micro(10⁻⁶)

BRIEF DESCRIPTION OF THE DRAWINGS

FIG. 1 is a plot showing the relative decrease in absorption uponexposure to light for selected dyes, as set forth in Example 15.

FIG. 2 is a plot showing the relative decrease in fluorescence intensityupon exposure to light for selected dyes, as set forth in Example 15.

DETAILED DESCRIPTION OF THE INVENTION

The invention relates generally to dyes and photoluminescent compoundscontaining at least one alkyl-phosphonate or substitutedalkyl-phosphonate group including their synthetic precursors, and tomethods of synthesizing and using such compounds. These compounds may beuseful in both free and conjugated forms, as probes, labels, and/orindicators. This usefulness may reflect in part an enhancement of one ormore of the following: quantum yield, Stokes' shift, extinctioncoefficients, aqueous solubility, photostability and chemical stability.Further specific derivatives of these esters can function as“biologically active” fluorescent compounds that may passively penetratethe plasma membranes of cells, where an intracellular conversion of thecompounds into cell-impermeant forms result in the converted compoundsremaining trapped within the cell membranes.

In one aspect, the invention relates to compositions that includereporter compounds that include or are substituted by at least onealkyl-phosphonate or substituted alkyl-phosphonate group.

These alkyl-phosphonate residues may either be neutral substituents thatdo not add additional formal charges to the overall molecule:—(CH₂)_(n)—PO₃Et₂ or —(CH₂)_(n)—PO₃[(CH₂)_(n)COOR]₂, R=Me, Et or—(CH₂)_(n)—PO₃[(CH₂)_(n)OH]₂, —(CH₂)_(n)—PO₃[CH₂O(C═O)CH₃]₂, and/or theymay include functionalities with reactive,—(CH₂)_(n)—PO₃[(CH₂)_(n)COONHS]₂, ionic —(CH₂)_(n)—PO₃[(CH₂)_(n)SO₃ ⁻]₂and carrier groups —(CH₂)_(n)—PO₃[(CH₂)_(n)CO—NH—S_(c)]₂.

As shown in Example 15, the introduction of alkyl-phosphonate groups,such as —(CH₂)₂—PO₃Et₂, into cyanines may substantially increase theirphotostability up to twice as high as those of conventional cyanines,e.g. Cy5, a cyanine containing only sulfogroups.

This property may help produce dyes with higher photostability andexcitation and emission in relatively inaccessible regions of thespectrum, including the red and near infrared.

The alkyl-phosphonate [R—(CH₂)_(n)]— group in the structure above servesmultiple purposes: either it may be used to help increase thephotostability of a cyanine dye R═CH₃, n=1 (see above). Further theethyl-phosphonate group can be easily used as a convenient startingfunctional group to introduce other residues (see Example 2), e.g.reactive groups (R═(CH₂)_(n)COOH, (CH₂)_(n)COO—NHS), ionic/hydrophilicgroups (R═(CH₂)_(n)SO₃ ^(⊖), (CH₂)_(n)OH) or linked carriers(R═(CH₂)_(n)CO—NH—S_(c)).

As each phosphonate has 2 modifiable alkyl-substituents, thesubstitution with ionic groups such as e.g. sulfo-alkyl as described in1t or 1u (Example 2), may provide a highly negatively chargedfunctionality —(CH₂)_(n)—PO₃[(CH₂)_(n)SO₃ ⁻]₂ that may improve thewater-solubility of the final cyanine dye. Alternatively or in addition,the branched nature of these substituents (see Example 16, laststructure and below) may also help in reducing the tendency of thecompounds to form aggregates.

Ethyl-phosphonates can also be converted to “biologically activegroups”, which as outlined in an article by C. Schultz in Bioorganic &Medicinal Chemistry 11, 885-898, (2003) open up ways to producenon-charged compounds that are able to passively diffuse across theplasma membrane. Once inside the cells, the masking group, e.g. anaceteoxymethylene group, (CH₂)_(n)—PO₃[CH₂O(C═O)CH₃]₂ is removed bychemical or enzymatic hydrolysis, generating the charged phosphate orphosphonate ester, and thereby making the dye-molecule again impermeantto cell membranes and thereby preventing dye leakage from the cell.

The synthesis of these phosphate esters is described by D. N. Srivastvaand D. Farquhar, Bioorg. Chem. 1984, 12, 118 and typically proceeds viaan acyloxyalkyl halogenide, in the presence of a sterically hinderedbase, in a dry organic solvent.

The remaining discussion includes (1) an overview of structures, (2) anoverview of synthetic methods, and (3) a discussion of the applicationsof the invention.

Overview of Structures

The reporter compounds are typically polymethine dyes, such as cyaninedyes, merocyanine dyes, and styryl dyes. These dyes are characterized inthat they typically include at least one of the following heterocyclicmoieties:

where X⁻ represents an anion;

R¹ is selected from H, L-S_(c), L-Rx, L-R^(x), —CH₂—CONH—SO₂-Me, L-PO₃^(2⊖), L-O—PO₃ ^(2⊖), L-PO₃R^(m⊖), L-O—PO₃R^(m⊖), aliphatic groups,alicyclic groups, alkylaryl groups, aromatic groups, each aliphaticresidue may incorporate up to six heteroatoms selected from N, O, S, andcan be substituted one or more times by F, Cl, Br, I, hydroxy, alkoxy,carboxy, sulfo, phosphate, amino, sulfate, phosphonate, cyano, nitro,azido, alkyl-amino, dialkyl-amino or trialkylammonium; and

L is a single covalent bond or is a covalent linkage that is linear orbranched, cyclic or heterocyclic, saturated or unsaturated, having 1-20nonhydrogen atoms from the group of C, N, P, O and S, in such a way thatthe linkage contains any combination of ether, thioether, amine, ester,amide bonds; single, double, triple or aromatic carbon-carbon bonds; orcarbon-sulfur bonds, carbon-nitrogen bonds, phosphorus-sulfur,nitrogen-nitrogen, nitrogen-oxygen or nitrogen-platinum bonds, oraromatic or heteroaromatic bonds;

R^(x) is a reactive group;

S_(c) is a conjugated substance;

R^(±) is an ionic group;

R^(a) may be, H, L-S_(c), L-R^(x), L-R^(±), —CH₂—CONH—SO₂-Me, aliphatic,alicyclic, aromatic, alkyl-aryl, F, Cl, Br, I, NH₂, —COOH, —CN, azido,—OH, —NO₂, —SO₃H, —SO₂NHR^(m), —SO₂NHNH—R^(m), —SO₂R^(l), —C₆H₄—SO₃^(⊖), —C₆H₄—PO₃ ^(⊖), pyridylium, pyrylium, —PO₃ ^(2⊖), —O—PO₃ ^(2⊖),—PO₃R^(m⊖), —O—PO₃R^(m⊖), —CONH₂, CONHR^(m), —CONHNHR^(m), COO—NHS andCOO—R^(m);

(R³)^(±) is selected from L-PO₃R^(m⊖), L-PO₃R^(m) ₂;

R^(m) is selected from a group consisting of L-S_(c), L-R^(x), L-R^(±),aliphatic substituents, aromatic substituents; each aliphatic residuemay incorporate up to six heteroatoms selected from N, O, S, and can besubstituted one or more times by F, Cl, Br, I, hydroxy, alkoxy, carboxy,sulfo, phosphate, amino, sulfate, phosphonate, cyano, nitro, azido,alkyl-amino, dialkyl-amino or trialkylammonium;

R^(l) is selected from alkyl, —CH₂F, CHF₂, CF₃;

each of X¹, X², X³, and X⁴ are independently selected from the groupconsisting of N, NR^(ι), O, S, and C—R^(ι), wherein R^(ι) is hydrogen,L-S_(c), L-R^(x), L-R^(±), —CH₂—CONH—SO₂-Me, L-PO₃R^(n⊖), L-PO₃R₂ ^(n),or an aliphatic, alicyclic, or aromatic group; amino, sulfo,trifluoromethyl, alkoxy, halogen, carboxy, hydroxy, phosphate,phosphonate, sulfate; or adjacent R^(ι) substituents, taken incombination, form a fused aromatic or heterocyclic ring that is itselfoptionally further substituted by H, L-S_(c), L-R^(x), L-R^(±), alkyl,aryl or cycloalkyl.

R^(n) is selected from a group consisting of L-S_(c), L-R^(x), L-R^(±).

The substituents on the substituted rings may be chosen quite broadly,and may include the various component listed above, among others.

in one aspect of the invention, the reporter compounds are described bythe following structure:

Here, Z is a single carbon-center, a partially-conjugated four- five- orsix-member ring-system, and A, B, and C are substituents of Z; Z is asingle carbon-center or part of a partially-conjugated four- five orsix-member ring system, and A is represented by R^(τ) and B or C is oneof W¹ and W²

The components R¹, R^(η), R^(τ), n, m, X¹, X², X³, X⁴, and Y are definedin detail in the Detailed Description. However, generally, each compoundincludes at least one of W¹ or W² with the preferred syntheticprecursors including one, and the preferred reporter compounds includingtwo. Alternatively, or in addition, the compound may include at leastone heteroatom in X¹ through X⁴ of W¹, W² or W³. Alternatively, or inaddition, the compound may include a reactive group and/or a carrier.

Reporter Compounds

The reporter compounds may be colorimetric dyes, useful as stains andfor colorimetric detection. Alternatively or in addition, the reportercompounds may be photoluminescent, particularly fluorescent, and mayhave utility in photoluminescence assays and methods, as discussedabove. All compounds are characterized that they contain at least onephosphonate or substituted phosphonate group in the heterocyclic base.

Tandems

Reporter compounds in accordance with the invention also may includepairs, triplets, and higher numbers of compounds conjugated together toform a single compound. Such “tandems” may be used to obtain alternativespectral properties, such as enhanced Stokes' shifts. Such tandems maybe based on the principle of energy transfer. Some potentialcombinations are drawn below, where B and C, W¹, W² and Z have theirusual meanings, and U represents a cross-link, such as may be formed bycross-reaction using a reactive compound. U can be also be chosen sothat the two pairs become a larger conjugated system linked via aC—C-triple-bond as described by K. Burgess, Chem. Eur. J. 2003, 9,4430-4441. Z and each substituent may be chosen independently for eachcomponent of a tandem.

Reactive Groups R^(x)

The substituents of Z may include one or more reactive groups, where areactive group generally is a group capable of forming a covalentattachment with another molecule or substrate. Such other molecules orsubstrates may include proteins, carbohydrates, nucleic acids, andplastics, among others. Reactive groups vary in their specificity, andmay preferentially react with particular functionalities and moleculetypes. Thus, reactive compounds generally include reactive groups chosenpreferentially to react with functionalities found on the molecule orsubstrate with which the reactive compound is intended to react.

The compounds of the invention are optionally substituted, eitherdirectly or via a substituent, by one or more chemically reactivefunctional groups that may be useful for covalently attaching thecompound to a desired substance. Each reactive group, or R^(x), may bebound to the compound directly by a single covalent bond, or may beattached via a covalent spacer or linkage, L, and may be depicted as-L-R^(x).

The reactive functional group of the invention R^(x) may be selectedfrom the following functionalities, among others: activated carboxylicesters, acyl azides, acyl halides, acyl halides, acyl nitriles, acylnitriles, aldehydes, ketones, alkyl halides, alkyl sulfonates,anhydrides, aryl halides, aziridines, boronates, carboxylic acids,carbodiimides, diazoalkanes, epoxides, haloacetamides, halotriazines,imido esters, isocyanates, isothiocyanates, maleimides,phosphoramidites, silyl halides, sulfonate esters, and sulfonyl halides.

In particular, the following reactive functional groups, among others,are particularly useful for the preparation of labeled molecules orsubstances, and are therefore suitable reactive functional groups forthe purposes of the reporter compounds:

-   a) N-hydroxysuccinimide esters, isothiocyanates, and    sulfonylchlorides, which form stable covalent bonds with amines,    including amines in proteins and amine-modified nucleic acids;-   b) Iodoacetamides and maleimides, which form covalent bonds with    thiol-functions, as in proteins;-   c) Carboxyl functions and various derivatives, including    N-hydroxybenztriazole esters, thioesters, p-nitrophenyl esters,    alkyl, alkenyl, alkynyl, and aromatic esters, and acyl imidazoles;-   d) Alkylhalides, including iodoacetamides and chloroacetamides;-   e) Hydroxyl groups, which can be converted into esters, ethers, and    aldehydes;-   f) Aldehydes and ketones and various derivatives, including    hydrazones, oximes, and semicarbozones;-   g) Isocyanates, which may react with amines;-   h) Activated C═C double-bond-containing groups, which may react in a    Diels-Alder reaction to form stable ring systems under mild    conditions;-   i) Thiol groups, which may form disulfide bonds and react with    alkylhalides (such as iodoacetamide);-   j) Alkenes, which can undergo a Michael addition with thiols, e.g.,    maleimide reactions with thiols;-   k) Phosphoramidites, which can be used for direct labeling of    nucleosides, nucleotides, and oligonucleotides, including primers on    solid or semi-solid supports;-   l) Primary amines that may be coupled to variety of groups including    carboxyl, aldehydes, ketones, and acid chlorides, among others;-   m) Boronic acid derivatives that may react with sugars-   n) Pyrylium moieties react with primary amines.-   o) Haloplatinates form stable platinum complexes with amines, thiols    and heterocycles-   p) Aryl halides react with thiols and amines    R Groups

The R moieties associated with the various substituents of Z may includeany of a number of groups, as described above, including but not limitedto alicyclic groups, aliphatic groups, aromatic groups, and heterocyclicrings, as well as substituted versions thereof.

Aliphatic groups may include groups of organic compounds characterizedby straight- or branched-chain arrangement of the constituent carbonatoms. Aliphatic hydrocarbons comprise three subgroups: (1) paraffins(alkanes), which are saturated and comparatively unreactive; (2) olefins(alkenes or alkadienes), which are unsaturated and quite reactive; and(3) acetylenes (alkynes), which contain a triple bond and are highlyreactive. In complex structures, the chains may be branched orcross-linked and may contain one or more heteroatoms (such as polyethersand polyamines, among others).

As used herein, “alicyclic groups” include hydrocarbon substituents thatincorporate closed rings. Alicyclic substituents may include rings inboat conformations, chair conformations, or resemble bird cages. Mostalicyclic groups are derived from petroleum or coal tar, and many can besynthesized by various methods. Alicyclic groups may optionally includeheteroalicyclic groups that include one or more heteroatoms, typicallynitrogen, oxygen, or sulfur. These compounds have properties resemblingthose of aliphatics and should not be confused with aromatic compoundshaving the hexagonal benzene ring. Alicyclics may comprise threesubgroups: (1) cycloparaffins (saturated), (2) cycloolefins (unsaturatedwith two or more double bonds), and (3) cycloacetylenes (cyclynes) witha triple bond. The best-known cycloparaffins (sometimes callednaphthenes) are cyclopropane, cyclohexane, and cyclopentane; typical ofthe cycloolefins are cyclopentadiene and cyclooctatetraene. Mostalicyclics are derived from petroleum or coal tar, and many can besynthesized by various methods.

Aromatic groups may include groups of unsaturated cyclic hydrocarbonscontaining one or more rings. A typical aromatic group is benzene, whichhas a 6-carbon ring formally containing three double bonds in adelocalized ring system. Aromatic groups may be highly reactive andchemically versatile. Most aromatics are derived from petroleum and coaltar. Heterocyclic rings include closed-ring structures, usually ofeither 5 or 6 members, in which one or more of the atoms in the ring isan element other than carbon, e.g., sulfur, nitrogen, etc. Examplesinclude pyridine, pyrole, furan, thiophene, and purine. Some 5-memberedheterocyclic compounds exhibit aromaticity, such as furans andthiophenes, among others, and are analogous to aromatic compounds inreactivity and properties.

Any substituent of the compounds of the invention, including anyaliphatic, alicyclic, or aromatic group, may be further substituted oneor more times by any of a variety of substituents, including withoutlimitation, F, Cl, Br, I, carboxylic acid, sulfonic acid, CN, nitro,hydroxy, phosphate, phosphonate, sulfate, cyano, azido, amine, alkyl,alkoxy, trialkylammonium or aryl. Aliphatic residues can incorporate upto six heteroatoms selected from N, O, S. Alkyl substituents includehydrocarbon chains having 1-22 carbons, more typically having 1-6carbons, sometimes called “lower alkyl”.

As described in WO01/11370, sulfonamide groups such as—(CH₂)_(n)—SO₂—NH—SO₂—R, —(CH₂)_(n)—CONH—SO₂—R, —(CH₂)_(n)—SO₂—NH—CO—R,and —(CH₂)_(n)—SO₂NH—SO₃H, where R is aryl or alkyl and n=1-6, can beused to reduce the aggregation tendency and have positive effects on thephotophysical properties of cyanines and related dyes, in particularwhen these functionalities are directly associated with the benzazolering in position 1 (the nitrogen atom in the azole ring).

Where a substituent R is further substituted by a functional group thatis formally electronically charged, such as for example a carboxylicacid, sulfonic acid, phosphoric acid, phosphonate or a quaternaryammonium group, the resulting ionic substituent R^(±) may serve toincrease the overall hydrophilicity of the compound. Examples ofelectronically charged functional groups include —PO₃ ^(2⊖), —O—PO₃^(2⊖), —PO₃R^(m⊖), —O—PO₃R^(m⊖), —C₆H₄—SO₃ ^(⊖), —C₆H₄—PO₃ ^(⊖),pyridylium, pyrylium, —SO₃ ^(⊖), —O—SO₃ ^(⊖), —COO^(⊖) and ammonium,among others.

As used herein, functional groups such as “carboxylic acid,” “sulfonicacid,” and “phosphoric acid” include the free acid moiety as well as thecorresponding metal salts of the acid moiety, and any of a variety ofesters or amides of the acid moiety, including without limitation alkylesters, aryl esters, and esters that are cleavable by intracellularesterase enzymes, such as alpha-acyloxyalkyl ester (for exampleacetoxymethylene esters, among others). Further these esters mightcontain additional reactive or ionic groups and linked carriers.

The compounds of the invention are optionally further substituted by areactive functional group R^(x), or a conjugated substance S_(c), asdescribed below.

The compounds of the invention may be depicted in structuraldescriptions as possessing an overall charge, it is to be understoodthat the compounds depicted include an appropriate counter ion orcounter ions to balance the formal charge present on the compound.Further, the exchange of counter ions is well known in the art andreadily accomplished by a variety of methods, including ion-exchangechromatography and selective precipitation, among others.

Carriers and Conjugated Substances S_(c)

The reporter compounds of the invention, including synthetic precursorcompounds, may be covalently or non-covalently associated with one ormore substances. Covalent association may occur through variousmechanisms, including a reactive functional group as described above,and may involve a covalent linkage, L, separating the compound orprecursor from the associated substance (which may therefore be referredto as -L-S_(c)).

The covalent linkage L binds the reactive group R^(x), the conjugatedsubstance S_(c) or the ionic group R^(±) to the dye molecule, eitherdirectly (L is a single bond) or with a combination of stable chemicalbonds, that include single, double, triple or aromatic carbon-carbonbonds; carbon-sulfur bonds, carbon-nitrogen bonds, phosphorus-sulfurbonds, nitrogen-nitrogen bonds, nitrogen-oxygen or nitrogen-platinumbonds, or aromatic or heteroaromatic bonds; L includes ether, thioether,carboxamide, sulfonamide, urea, urethane or hydrazine moieties.Preferable L include a combination of single carbon-carbon bonds andcarboxamide or thioether bonds.

Where the substance is associated noncovalently, the association mayoccur through various mechanisms, including incorporation of thecompound or precursor into or onto a solid or semisolid matrix, such asa bead or a surface, or by nonspecific interactions, such as hydrogenbonding, ionic bonding, or hydrophobic interactions (such as Van derWaals forces). The associated carrier may be selected from the groupconsisting of polypeptides, polynucleotides, polysaccharides, beads,microplate well surfaces, metal surfaces, semiconductor andnon-conducting surfaces, nano-particles, and other solid surfaces.

The associated or conjugated substance may be associated with orconjugated to more than one reporter compound, which may be the same ordifferent. Generally, methods for the preparation of dye-conjugates ofbiological substances are well-known in the art. See, for example,Haugland et al., MOLECULAR PROBES HANDBOOK OF FLUORESCENT PROBES ANDRESEARCH CHEMICALS, Eighth Edition (1996), which is hereby incorporatedby reference. Typically, the association or conjugation of a chromophoreor luminophore to a substance imparts the spectral properties of thechromophore or luminophore to that substance.

Useful substances for preparing conjugates according to the presentinvention include, but are not limited to, amino acids, peptides,proteins, nucleosides, nucleotides, nucleic acids, carbohydrates,lipids, ion-chelators, nonbiological polymers, cells, and cellularcomponents. The substance to be conjugated may be protected on one ormore functional groups in order to facilitate the conjugation, or toinsure subsequent reactivity.

Where the substance is a peptide, the peptide may be a dipeptide orlarger, and typically includes 5 to 36 amino acids. Where the conjugatedsubstance is a protein, it may be an enzyme, an antibody, lectin,protein A, protein G, hormones, or a phycobiliprotein. The conjugatedsubstance may be a nucleic acid polymer, such as for example DNAoligonucleotides, RNA oligonucleotides (or hybrids thereof), orsingle-stranded, double-stranded, triple-stranded, or quadruple-strandedDNA, or single-stranded or double-stranded RNA.

Another class of carriers includes carbohydrates that arepolysaccharides, such as dextran, heparin, glycogen, starch andcellulose.

Where the substance is an ion chelator, the resulting conjugate may beuseful as an ion indicator (calcium, sodium, magnesium, zinc, potassiumand other important metal ions) particularly where the opticalproperties of the reporter-conjugate are altered by binding a targetion. Preferred ion-complexing moieties are crown ethers (U.S. Pat. No.5,405,957) and BAPTA chelators (U.S. Pat. No. 5,453,517).

The associated or conjugated substance may be a member of a specificbinding pair, and therefore useful as a probe for the complementarymember of that specific binding pair, each specific binding pair memberhaving an area on the surface or in a cavity which specifically binds toand is complementary with a particular spatial and polar organization ofthe other. The conjugate of a specific binding pair member may be usefulfor detecting and optionally quantifying the presence of thecomplementary specific binding pair member in a sample, by methods thatare well known in the art.

Representative specific binding pairs may include ligands and receptors,and may include but are not limited to the following pairs:antigen-antibody, biotin-avidin, biotin-streptavidin, IgG-protein A,IgG-protein G, carbohydrate-lectin, enzyme-enzyme substrate;ion-ion-chelator, hormone-hormone receptor, protein-protein receptor,drug-drug receptor, DNA-antisense DNA, and RNA-antisense RNA.

Preferably, the associated or conjugated substance includes proteins,carbohydrates, nucleic acids, and nonbiological polymers such asplastics, metallic nanoparticles such as gold, silver and carbonnanostructures among others. Further carrier systems include cellularsystems (animal cells, plant cells, bacteria). Reactive dyes can be usedto label groups at the cell surface, in cell membranes, organelles, orthe cytoplasm.

Finally these compounds can be linked to small molecules such as aminoacids, vitamines, drugs, haptens, toxins, environmental pollutants.Another important ligand is tyramine, where the conjugate is useful as asubstrate for horseradish peroxidase. Additional embodiments aredescribed in U.S. Patent Application Publication No. US 2002/0077487.

Synthesis

The synthesis of the disclosed reporter compounds typically is achievedin a multi-step reaction, starting with the synthesis of a methylenebase. The synthesis of suitable methylene bases may proceed based onliterature or novel methods. Generally, the spectral properties of thereporter compounds, including excitation and emission wavelengths forluminescent compounds, may be strongly dependent on the type ofmethylene base used. Typical starting materials include quarternizedindolenines, benzthiazoles, benzoxazoles, benzimidazoles, etc.,N,N′-diphenylformamidine, and malonaldehyde bis(phenylimine)monohydrochloride, among others.

The majority of current indolenine-cyanine labels are either based onindolenines that are exclusively 3,3′-dimethyl indolenines (Mujumdar etal., Bioconjugate Chem. 4(2) 105-111, 1993; U.S. Pat. No. 4,981,977,Southwick et al.; U.S. Pat. No. 5,268,486 Waggoner et al.; U.S. Pat. No.5,486,616 Waggoner et al.) or 3-methyl-3′-alkyl indolenines wherein thealkyl linker is either modified to contain a reactive C₆-linker (U.S.Pat. No. 6,258,340; Licha et al. and U.S. Pat. No. 6,977,305; Leung etal.) or a sulfo-group with a C₃ or C₄-linker (U.S. Pat. No. 6,258,340;Licha et al. and US patent application 2007/0128659A1; Czerney et al).Compounds with shorter linkers to these sulfo-groups are claimed buthave not been reduced to practice.

One of the preferred ways of synthesis of these novel methylene-bases isvia a “Fischer-Indole Synthesis” using substituted phenyl-hydrazines andsubstituted aliphatic ketones:

Starting materials like ethyl 2-methylacetoacetate are also readilyavailable from Aldrich. The reaction of this starting material with4-hydrazinobenzenesulfonic acid according to the procedure of K. Liu etal., Org. Lett., 2006, 8 (25), 5769-5771 is described below.

The new indolenine intermediates, synthesized via the “Fischer-Indolesynthesis” (see above) are subsequently quarternized with alkylatingagents such as methyl iodide, propanesultone, butanesultone orbromo-hexanoic acid. The synthesis of other key-compounds for thesenovel cyanines are described in Examples 1 and 2, while the methods forthe synthesis of representative dyes of this invention are provided inExamples 3-12.

The dye molecules of this invention typically consist of a bridging unitand the heterocyclic bases W¹ and W². The bridging unit is either asimple polymethine chain of various lengths or it can be substituted bya cyclo-alkene. When the bridging unit is a polymethine chain thecoupling agent can be N,N-diphenylformamidine, triethylorthoformate, ormalonaldehyde bis(phenylimine) hydrochloride, 1,1,3-trimethoxypropane,1,1,3,3-tetramethoxypropane and glutaconaldehyde dianilmonohydrochloride.

The synthesis of various classes of polymethine dyes is very welldescribed in the book by Gupta RR, Strekowski L (eds) (2008)Heterocyclic polymethine dyes. Topics in Heterocyclic Chemistry, vol.14. Springer-Verlag, Berlin, Heidelberg. Other resources are A. Mishraet al., Cyanines during the 1990s: a review. Chem. Rev. 100, 1973-2011(2000), and Gonçalves et al., Fluorescent labeling of biomolecules withorganic probes. Chem. Rev. 109, 190-212, (2009).

To further enhance water-solubility, sulfonic acid or other groupsincluding quaternary ammonium, polyether, carboxyl, and phosphate, amongothers, may be introduced into the heterocyclic ring systems or in thebridging unit. In order to facilitate covalent attachment to proteins,reactive N-hydroxy-succinimide ester (NHS ester) or other reactivederivatives may be synthesized.

The synthesis of cyanine dyes is described in Mujumdar et al.,Bioconjugate Chem. 4(2) 105-111, 1993 and in several other patentapplications (U.S. Patent Appl. US 2002/0077487 A1, U.S. patentapplication US2003/0170179, U.S. Pat. No. 5,569,587, U.S. Pat. No.5,672,027, U.S. Pat. No. 5,808,044, U.S. patent applicationUS2006/0280688, U.S. patent application US2004/0014981, and WO2004/039894, which are incorporated herein as reference).

The cyanine dyes of this invention exhibit absorption maxima in therange between 500 and 850 nm. In addition to a variety of otherstructural parameters, the selection of a monomethine, trimethine, orpentamethine linkages permits the spectral properties of the resultingcompound to be altered according to the characteristics desired. Forexample, where the remainder of the compound is held constant, shiftingfrom a monomethine to a trimethine, pentamethine or heptamethine linkagein a W¹ or W² substituent typically results in a shifting of theabsorption and emission wavelengths of the resulting compounds toprogressively longer wavelengths. The absorption maxima can befine-tuned by additional introduction of functional groups to match theemission lines of a frequency-doubled Nd-Yag laser (532 nm), Kr-ionlaser (568 and 647 nm), the HeNe laser (543 nm and 633 nm) and diodelasers (635 nm, 650 nm, 780 nm etc.). Cyanine dyes exhibit a lessertendency to change their quantum yields upon changing the environment(e.g. labelling to a protein).

Many compounds of the invention possess an overall electronic charge. Itis to be understood that when such electronic charges are present, thatthey are balanced by an appropriate counterion, which may or may not beidentified.

EXAMPLE 1 Synthesis of Precursors and Intermediates

This section describes the synthesis of various precursors andintermediates for the synthesis of novel cyanine dyes.p-hydrazinobenzenesulfonic acid (Illy et al., J. Org. Chem. 33,4283-4285 1968), 2,3,3-trimethylindole-5-sulfonic acid potassium salt(1a), 1-(5-carboxypentyl)-2,3,3-trimethyl-3H-5-indoliumsulfonate (1b),1-(4-sulfonatobutyl)-2,3,3-trimethylindoleninium-5-sulfonate (1h)(Mujumdar et al., Bioconjugate Chem. 4(2) 105-111, 1993), and1,2,3,3-tetramethylindoleninium-5-sulfonate (1c) were synthesized usingliterature procedures. 1d-1f are synthesized according to the proceduresprovided in U.S. Patent Application Publication No. US 2002/0077487.1-(2-phosphonethyl)-2,3,3-trimethylindoleninium-5-sulfonate is describedin PCT Patent Application Publication No. WO 01/36973.

The synthesis of sulfonated benzindolenines and other cyclo-condensedheterocycles is described in U.S. Patent Application Publication No. US2002/0077487 and U.S. Pat. No. 6,140,494 and by S. Mujumdar et al.Bioconjugate Chem. 1996, 7, 356-362.

It is also understood that the additional aromatic ring can be fused atdifferent positions onto the parent heterocycle (see WO02/26891 A1) andMujumdar et al. Bioconjugate Chem. 1996, 7, 356-362.

The synthesis of heterocyclic compounds containing additionalheteroatoms is also described in U.S. Patent Application Publication No.US 2002/0077487.

Synthesis of p-hydrazinobenzenesulfonic acid

33 g of sodium carbonate was added to a suspension of 104 g (0.6 mol) ofp-aminobenzenesulfonic acid in 400 mL of hot water. The solution wascooled to 5° C. in an ice-bath, and 70 g of concentrated sulfuric acidwere added slowly under rapid stirring. A solution of 42 g of sodiumnitrite in 100 mL of water was then added under cooling. A light yellowdiazo-compound precipitate formed, which was filtered and washed withwater, but not dried.

The wet diazo-compound was added under stirring and cooling (5° C.) to asolution of 170 g of sodium sulfite in 500 mL of water. The solution,which turned orange, was stirred under cooling for 1 h, and then heatedto reflux. Finally, 400 mL of concentrated hydrochloric acid were added.The solution turned yellow, and the product precipitated as a whitesolid. For complete decoloration, 1-2 g of powdered zinc were added. Thereaction mixture was cooled overnight, and the precipitate was filtered,washed with water, and dried in an oven at 100° C.

Yield: 96 g (85%), white powder; M.P.=286° C. (Lit.=285° C.); R_(f):0.95 (RP-18, water:MeOH 2:1).

Synthesis of 2,3,3-trimethylindole-5-sulfonic acid, potassium salt (1a)

18.2 g (0.12 mol) of p-hydrazinobenzenesulfonic acid and 14.8 g (0.17mol) of isopropylmethylketone were stirred in 100 mL of glacial aceticacid at room temperature for 1 h. The mixture was then refluxed for 4 h.The mixture was cooled to room temperature, and the resulting pink solidprecipitate was filtered and washed with ether.

The precipitate was dissolved in methanol, and a concentrated solutionof potassium hydroxide in 2-propanol was added until a yellow solidcompletely precipitated. The precipitate was filtered, washed withether, and dried in a desiccator over P₂O₅.

Yield: 20.4 g (71%), off-white powder; M.P.=275° C.; R_(f): 0.40 (silicagel, isopropanol:water:ammonia 9:0.5:1).

1-(5-carboxypentyl)-2,3,3-trimethyl-3H-5-indoliumsulfonate (1b)

15.9 g (57 mmol) of 2,3,3-trimethylindolenium-5-sulfonic acid potassiumsalt 1a and 12.9 g (66 mmol) of 6-bromohexanoic acid were refluxed in100 mL of 1,2-dichlorobenzene for 15 min under a nitrogen atmosphere.The solution was cooled to room temperature, and the resulting pinkprecipitate was filtered, washed with chloroform, and dried.

Yield: 15.8 g (58%), pink powder; R_(f): 0.75 (RP-18, MeOH:water 2:1).

Synthesis of 1,2,3,3-tetramethylindolium-5-sulfonate (1c)

1.1 g of 2,3,3-trimethylindoleninium-5-sulfonate were suspended in 30 mLof methyl iodide. The reaction mixture was heated to boiling for h in asealed tube. After the mixture was cooled, excess methyl iodide wasdecanted, and the residue was suspended in 50 mL of acetone. Thesolution was filtered, and the residue was dried in a desiccator overCaCl₂. The resulting light yellow powder was used without furtherpurification.

Yield: 90%, light yellow powder.

Synthesis of 3-(5-carboxypentyl)-2,3-dimethyl-5-sulfo-1-(3-sulfopropyl)indolium sodium salt (1d), (Scheme I)

A mixture of 25 g of ethyl 2-methylacetoacetate (I), 64 ml of 21% sodiumethoxide solution in ethanol and 34 mL of ethyl-6-bromohexanoate isrefluxed in 200 mL of ethanol overnight. The mixture is filtered and thesolvent is removed under reduced pressure. The residue is partitionedbetween 1 M HCl and chloroform.

The organic layer is dried over magnesium sulfate and purified on silicagel using 1:10 ethyl acetate/hexane as the eluent to yield 22 g of ethyl2-(5-carboethoxypentyl)-2-methylactoacetate (IIa)

The above compound is dissolved in 300 ml of methanol. A solution of 10g NaOH in 100 mL water is added. The mixture is heated at 50° C.overnight. The solution is reduced to about 50 mL, acidified to pH 1 andextracted with ethyl acetate. The organic phase is collected, dried overMgSO₄ and evaporated to yield 13.5 g of 7-methyl-8-oxononanonic acid(IIIa).

The nonanonic acid is refluxed in 110 mL of acetic acid with 13.5 g of4-hydrazinobenzenesulfonic acid for 4 hours. The acetic acid isevaporated and the product is purified on silica gel to yield 23 g ofthe product (IVa).

To the methanol solution of 11 g of Compound IVa is added 3.4 g ofanhydrous sodium acetate. The mixture is stirred for five minutes. Thesolvent is evaporated and the resulting sodium salt is heated with 24.4g of propane sultone at 110° C. for 1 hour to generate the final product1d.

Synthesis of 3-(6-hydroxyhexyl)-2,3-dimethyl-5-sulfo-1-(3-sulfopropyl)indolium, sodium salt (1e)

Another starting material 1e is synthesized analogously using ethyl2-methylacetoacetate and 6-benzoyl-1-bromo-hexane in the presence of 1.2equivalents of sodium hydride in THF according to 1d. After isolatingthe 3-(6-hydroxyhexyl)-2,3-dimethyl-5-sulfo-indolium, inner salt (thehydroxy group is again protected and the compound is quarternized usingpropanesultone. Deprotection is achieved using dilute NaOH.

1f is synthesized analogously taking into account the more polar natureof the sulfonic groups that are introduced either by reaction with2-bromo-ethane-sulfonic acid, propane- or butanesultone. Sulfogroups canalso be introduced by reaction of a 3-carboxy-alkyl-substituted compoundlike 1d with taurine according to Terpetschnig et al. Anal. Biochem.217, 197-204 (1994).

Using 4-hydrazino-benzoic acid as described in Anal. Biochem. 217,197-204 (1994) or 4-hydrazino-phenyl-acetic acid as described inCytometry 11(3), 418-30 (1990) and reacting them in a Fischer indolesynthesis with 7-methyl-8-oxononanonic acid or one of the otherfunctionalized precursors as described above, 5-carboxy-derivatizedindoles such as 1 g that contain a spacer group in position 3 can besynthesized.

Other compounds that contain functional groups in both R³ and R⁴ can besynthesized as described below and used as starting materials forcyanine dyes of this invention. R³ and R⁴ can also be a part of analiphatic ring system as described in U.S. Patent ApplicationPublication No. US 2002/0077487.

Selected precursor compounds are shown below:

1

R³ 1 R¹ R² (y = 2, 3, 4) R⁴ a — SO₃ ⁻ CH₃ CH₃ b (CH₂)₅COOH SO₃K CH₃ CH₃c CH₃ SO₃ ⁻ CH₃ CH₃ d (CH₂)₃SO₃Na SO₃ ⁻ (CH₂)₅COOH CH₃ e (CH₂)₃SO₃Na SO₃⁻ (CH₂)₆OH CH₃ f (CH₂)₃SO₃Na SO₃ ⁻ (CH₂)_(y)SO₃Na CH₃ g (CH₂)₃SO₃ ⁻ COOH(CH₂)₅COOH CH₃ h (CH₂)₃SO₃Na SO₃ ⁻ CH₃ CH₃ i — SO₃ ⁻ (CH₂)₂PO(OEt)₂ CH₃

EXAMPLE 2 Novel Indolenine Intermediates Synthesis of3-(2-phosphonoethyl)-3H-5-indolenines2,3-dimethyl-3-(2-diethylphosphonatethyl)-3H-5-indolesulfonic acid (1i)

A mixture of 4.70 g (20 mmol) of diethyl3-methyl-4-oxo-1-butylphosphonate, 3.75 g (20 mmol) of4-hydrazinobenzenesulfonic acid (synthesis as described above), and 40ml of acetic acid was refluxed for 20 hours. The insoluble precipitatewas filtered and the filtrate was evaporated. The obtained residue wascolumn purified (RP-18, water) to yield 1.27 g (13.7%) of title productas brown sludge oil. λ_(abs) 260 nm (water). δ_(H) (200 MHz, DMSO-d₆):7.69 (1H, s, arom H), 7.61 (1H, d, 8.2 Hz, arom H), 7.41 (1H, d, 8.2 Hzarom H), 3.99-3.78 (4H, m, POCH ₂), 2.30 (3H, s, 2-CH ₃), 2.23-1.84 (2H,m, CH ₂) 1.33 (3H, s, 3-CH ₃), 1.24-1.09 (6H, m, CH₂ CH ₃), 0.96-0.71(2H, m, CH ₂).

Synthesis of diethyl [2-(2,3,5-trimethyl-3H-indol-3-yl)ethyl]phosphonate(1j)

A mixture of 320 mg (2.01 mmol) 3-methyl-4-oxo-1-butylphosphonate, 480mg (2.01 mmol) (4-methylphenyl)hydrazine hydrochloride and 7 ml aceticacid were refluxed for 15 hours. The solvent was removed under reducedpressure and residue was dried. The product was extracted with ether.Yield: 83%. λ_(abs) 275 nm (methanol). δ_(H) (200 MHz, DMSO-d₆): 7.31(1H, d, 8.1 Hz arom H), 7.20 (1H, s, arom H), 7.09 (1H, d, 8.1 Hz aromH), 4.03-3.75 (4H, m, POCH₂), 2.33 (3H, s, 5-CH₃), 2.15 (3H, s, 2-CH₃),2.07-1.83 (2H, m, CH₂), 1.24 (3H, s, 3-CH₃), 1.16 (6H, t, 7.1 Hz CH₃),0.90-0.55 (2H, m, CH₂).

Synthesis of diethyl [2-(2,3-dimethyl-3H-indol-3-yl)ethyl]phosphonate(1k)

A mixture of 920 mg (3.89 mmol) 3-methyl-4-oxo-1-butylphosphonate, 560mg (3.89 mmol) phenylhydrazine hydrochloride and 10 ml acetic acid wererefluxed for 5 hours. The solvent was removed under reduced pressure andresidue was dried. The product was extracted with ether. Yield: 80% 1k.λ_(abs) 257 nm (methanol). δ_(H) (200 MHz, DMSO-d₆): 7.48-7.15 (4H, m,arom H), 3.97-3.77 (4H, m, POCH₂), 2.18 (3H, s, 2-CH₃), 2.15-1.80 (2H,m, CH₂), 1.27 (3H, s, 3-CH₃), 1.16 (6H, t, 7.0 Hz CH₃), 1.06-0.56 (2H,m, CH₂).

Synthesis of potassium2,3-dimethyl-3-(2-phosphonoethyl)-3H-indole-5-sulfonate (1l)

2,3-dimethyl-3-(2-diethylphosphonatethyl)-3H-5-indolesulfonic acid 1i(390 mg, 1 mmol) was dissolved in 5 ml of hydrogen bromide. The mixturewas heated at reflux for 4 hours and then cooled to room temperature.Aqueous solution of potassium hydroxide was added slowly until pH 12.The solvent was removed under reduced pressure and residue was columnpurified (RP-18, water) to give the title product. Yield: 80 mg 1l(21%). λ_(abs) 260 nm (water). δ_(H) (200 MHz, DMSO-d₆): 7.62 (1H, s,arom H), 7.56 (1H, d, 8.2 Hz arom H), 7.32 (1H, d, 8.1 Hz arom H), 2.16(3H, s, 2-CH₃), 2.10-1.77 (2H, m, CH ₂), 1.20 (3H, s, 3-CH₃), 0.67-0.31(2H, m, CH ₂).

Synthesis ofdi(ethylcarboxypentyloate)[2-(2,3-dimethyl-3H-indol-3-yl)ethyl]phosphonate(1m)

A mixture of 40 mg (0.13 mmol) diethyl[2-(2,3-dimethyl-3H-indol-3-yl)ethyl]phosphonate (1k) and 60 mg (0.27mmol) ethyl 6-bromohexanoate were heated at 135° C. for 2 hours in argonatmosphere. The product was triturated with hexane, filtered and dried.λ_(abs) 278 nm (methanol). δ_(H) (200 MHz, DMSO-d₆): 7.30-6.54 (4H, m,arom H), 4.15-3.91 (4H, m, POCH₂), 3.92-3.62 (4H, m, COCH₂), 2.17 (3H,s, 2-CH₃), 2.26 (4H, t, 6.8 Hz CH₂COOEt), 1.68-1.42 (6H, m, CH₂), 1.51(3H, s, 3-CH₃), 1.42-1.53 (8H, m, CH₂), 1.23-1.08 (6H, m, CH₃),0.95-0.62 (2H, m, CH₂).

Synthesis ofdi(carboxypentyl)[2-(2,3-dimethyl-3H-indol-3-yl)ethyl]phosphonate (1n)

A mixture of 500 mg (1.62 mmol) diethyl[2-(2,3-dimethyl-3H-indol-3-yl)ethyl]phosphonate (1k) and 790 mg (4.04mmol) bromogexanoic acid were heated at 135° C. for 2 hours in an argonatmosphere. The product was triturated with ether, filtered and dried.Yield: 90% in. λ_(abs) 279 nm (methanol). δ_(H) (200 MHz, DMSO-d₆):7.26-6.45 (4H, m, arom H), 4.09-3.91 (4H, m, POCH₂), 2.19 (4H, t, 6.9 HzCH₂COOH), 2.16 (3H, s, 2-CH₃), 1.64-1.40 (6H, m, CH₂), 1.52 (3H, s,3-CH₃), 1.40-1.16 (8H, m, CH₂), 0.91-0.59 (2H, m, CH₂).

Synthesis of dicarboxypenthyl[2-(2,3,5-trimethyl-3H-indol-3-yl)ethyl]phosphonate (1o)

A mixture of 540 mg (1.67 mmol) diethyl[2-(2,3,5-trimethyl-3H-indol-3-yl)ethyl]phosphonate (1j) and 820 mg(4.17 mmol) bromogexanoic acid were heated at 135° C. for 2 hours inargon atmosphere. The product was triturated with ether, filtered anddried. Yield: 71% 1o. λ_(abs) 292 nm (methanol). S_(H) (200 MHz,DMSO-d₆): 7.40-6.90 (3H, m, arom H), 3.98 (4H, t, 5.9 Hz POCH₂),2.33-2.12 (10H, m, 5-CH₃, 2-CH₃, CH₂COOH), 1.64-1.39 (10H, m, CH₂), 1.52(3H, s, 3-CH₃), 1.39-1.09 (6H, m, CH₂).

Synthesis of Potassium2,3-dimethyl-3-(2-phosphorylethyl)-1-(4-sulfonatobutyl)-3H-5-indoliumsulfonate(1p)

Potassium 2,3-dimethyl-3-(2-phosphonoethyl)-3H-indole-5-sulfonate 1i(100 mg, 0.27 mmol), 1,4-butansulton (0.11 ml, 1.1 mmol) and 0.1 ml ofacetic acid were mixed and heated at 150° C. for 5 hours. The rawproduct was washed with acetone, filtered and dried. Yield: 120 mg (88%)1p. λ_(abs)=275 nm. S_(H) (200 MHz, DMSO-d₆): 8.00 (2H, broad s, aromH), 7.85 (1H, d, 7.7 Hz arom H), 4.47 (2H, t, 5.5 Hz, NCH ₂), 2.85 (3H,s, 2-CH₃), 2.55-2.36 (2H, m, CH ₂SO₃H), 2.11-1.87 (2H, m, CH ₂),1.83-1.67 (2H, m, CH ₂), 1.57 (3H, s, 3-CH₃), 1.67-1.49 (2H, m, CH ₂),1.01-0.68 (2H, m, CH ₂).

Synthesis of Potassium2,3-dimethyl-3-[2-(diethylphosphoryl)ethyl]-1-(4-sulfonatobutyl)-3H-5-indoliumsulfonate (1q)

A mixture of potassium3-[2-(diethoxyphosphoryl)ethyl]-2,3-dimethyl-3H-indole-5-sulfonate 1i(460 mg, 1.08 mmol) and 1,4-butansulton (0.27 ml, 2.7 mmol) was heatedat 140° C. for 4 hours. The raw product was triturated with hot2-propanol, filtered and dried. Then the product was column purified(RP-18, water). Yield: 60 mg (10%) 1q. λ_(abs) 276 nm (water). δ_(H)(200 MHz, DMSO-d₆): 8.04 (1H, s, arom H), 8.01 (1H, d, 7.5 Hz arom H),7.84 (1H, d, 8.0 Hz arom H), 4.48 (2H, t, 6.6 Hz, NCH ₂), 4.06-3.81 (4H,m, P(CH ₂CH₃)₂), 2.87 (3H, s, 2-CH₃), 2.49-2.33 (2H, m, CH ₂SO₃H),2.02-1.84 (2H, m, CH ₂), 1.84-1.66 (2H, m, CH ₂), 1.59 (3H, s, 3-CH₃),1.66-1.50 (2H, m, CH ₂), 1.26-1.12 (6H, m, P(CH₂CH ₃)₂), 0.97-0.74 (2H,m, CH ₂).

Synthesis of1-(2-oxyethyl)-2,3-dimethyl-3-[2-(diethylphosphoryl)ethyl])-3H-indole-5-sulfonate(1r)

A mixture of potassium3-[2-(diethoxyphosphoryl)ethyl]-2,3-dimethyl-3H-indole-5-sulfonate (300mg, 0.7 mmol) and 2-chloroethanol (140 mg, 1.75 mmol) was heated at 130°C. for 10 hours. The raw product was triturated with ether, filtered anddried. λ_(abs) 274 nm (water). δ_(H) (200 MHz, DMSO-d₆): 8.03 (1H, s,arom H), 7.97 (1H, d, 7.5 Hz arom H), 7.83 (1H, d, 8.0 Hz arom H),4.71-4.53 (2H, m, NCH ₂), 4.06-3.71 (4H, m, P(CH ₂CH₃)₂), 3.64-3.40 (2H,m, CH ₂), 2.84 (3H, s, 2-CH₃), 1.58 (3H, s, 3-CH₃), 1.64-1.35 (2H, m, CH₂), 1.30-1.01 (6H, m, P(CH₂CH ₃)₂), 1.06-1.64 (2H, m, CH ₂).

Synthesis of dicarboxypenthyl[2-(1-(5-carboxypentyl)-2,3-dimethyl-3H-indoli-3-um)ethyl]phosphonatebromide (1s)

A mixture of 40 mg (0.083 mmol) dicarboxypenthyl[2-(2,3-dimethyl-3H-indol-3-yl)ethyl]phosphonate (1n) and 110 mg (0.166mmol) bromogexanoic acid were heated at 135° C. for 2 hours in an argonatmosphere. The product was triturated with ether, filtered and dried.λ_(abs) 278 nm (methanol). δ_(H) (200 MHz, DMSO-d₆): 8.08-7.56 (4H, m,arom H), 4.60-4.29 (2H, m, NCH ₂), 4.06-3.81 (4H, m, POCH₂), 2.84 (3H,s, 2-CH₃), 2.36-2.10 (6H, m, CH₂COOH), 1.93-1.68 (4H, m, CH₂), 1.68-1.15(16H, m, CH₂), 1.52 (3H, s, 3-CH₃), 0.91-0.52 (2H, m, CH₂).

Synthesis of Potassium sulfoethyl[2-(1-(2-sulfoethyl)-2,3-dimethyl-3H-indoli-3-um)ethyl]phosphonatebromide (1t)

A mixture of indolenine (200 mg, 0.65 mmol) 1k and 2-bromoethansulfonicacid (290 mg, 1.30 mmol) was heated at 150° C. in argon atmosphere for 2hours. The product was triturated with ether, filtered and dried. Yield:70%. λ_(abs) 275 nm (water). ¹H NMR (200 MHz, DMSO-d₆, ppm): δ 8.10-7.51(4H, m, arom H), 4.62-4.40 (2H, m, NCH₂), 4.14-3.92 (2H, m, POCH₂), 2.86(3H, s, 2-CH₃), 2.45-2.13 (4H, m, CH₂SO₃), 1.64-1.32 (2H, m, CH₂), 1.52(3H, s, 3-CH₃), 0.92-0.60 (2H, m, CH₂).

Synthesis of Potassium disulfoethyl[2-(1-(2-sulfoethyl)-2,3-dimethyl-3H-indoli-3-um)ethyl]phosphonatebromide (1u)

In the same way as above 200 mg (0.65 mmol) of indolenine 1k and2-bromoethansulfonic acid (440 mg, 1.95 mmol) was heated at 150° C. inargon atmosphere for 2 hours. The product was triturated with ether,filtered and dried. λ_(abs) 274 nm (water).

Synthesis of diethyl[2-(1-ethyl-2,3-dimethyl-3H-indoli-3-um)ethyl]phosphonate iodide (1v)

A mixture of 300 mg (0.97 mmol) diethyl[2-(2,3-dimethyl-3H-indol-3-yl)ethyl]phosphonate (1k), 0.5 ml ethyliodide and 5 ml acetonitryl were heated at 90° C. for 10 hours. Thesolvent was removed under reduced pressure and residue was dried. Yield:90% 1v. λ_(abs) 279 nm (methanol). δ_(H) (200 MHz, DMSO-d₆): 8.07-7.61(4H, m, arom H), 4.51 (2H, q 7.1, 14.5 Hz NCH₂), 4.06-3.74 (4H, m,POCH₂), 2.87 (3H, s, 2-CH₃), 2.46-2.20 (2H, m, CH₂), 1.58 (3H, s,3-CH₃), 1.45 (3H, t, 7.2 Hz CH₂CH ₃), 1.24-1.11 (6H, m, PO(CH₂CH ₃)₂),0.95-0.72 (2H, m, CH₂).

Synthesis of1-(5-carboxypentyl)-3,3-dimethyl-2-[(1E,3E)-4-(phenylamino)buta-1,3-dien-1-yl]-3H-indoliumbromide

A mixture of indolenine derivative 1x (500 mg, 1.41 mmol),malondialdehyde-bis-(phenylimin) monohydrochloride (550 mg, 2.11 mmol),5 ml of acetic acid and 5 ml of acetic anhydride were heated at refluxfor 4 hours. The solvent was removed under reduced pressure and residuewas triturated with ether. Yield: quantitative. λ_(abs) 446 nm(methanol).

Synthesis of1-(5-carboxyypentyl)-3,3-dimethyl-2-[(E)-2-(phenylamino)ethenyl]-3H-indolium-5-sulfonate

A mixture of indolenine derivative 1b, N,N′-diphenylimidoformamide andmethanol were heated at reflux for 8 hours. The solvent was removedunder reduced pressure until dry and residue was triturated with ethylacetate. The product was filtrated and dried. Yield: 90%. λ_(abs) 415 nm(water).

Synthesis of dipotassium2,3-dimethyl-1,3-di(4-sulfonatobutyl)-3H-5-indolium sulfonate (1y)Synthesis of 5-ethyloxycarbonyl-5-methyl-6-oxo-1-heptanesulfonic acid

0.35 ml (3.47 mmol) of 1,4-butane sultone were added to a mixture of0.49 ml (3.47 mmol) ethyl 2-methylacetoacetate and 513 mg (4.2 mmol) oftert-BuOK in 12 ml tert-butanol and refluxed for 15 hours. The formedprecipitate was filtered and washed with hexane to yield 730 mg of5-ethyloxycarbonyl-5-methyl-6-oxo-1-heptanesulfonic acid. δ_(H) (200MHz, DMSO-d₆): 4.12 (2H, q, 7.1, 14.2 Hz, OCH ₂), 3.97 (2H, t, 5.8 Hz,CH ₂SO₃H), 2.10 (3H, s, COCH ₃), 1.78-1.41 (4H, m, CH ₂), 1.24-1.01 (2H,m, CH ₂), 1.22 (3H, s, CCH ₃), 1.17 (3H, t, 7.3 Hz, CH₂ CH ₃).

Synthesis of 2,3-dimethyl-3-(4-sulfobutyl)-3H-5-indolesulfonic acid(IVd)

A solution of 290 mg of NaOH in 3 ml water was added to the mixture of710 mg (2.53 mmol) of5-ethyloxycarbonyl-5-methyl-6-oxo-1-heptanesulfonic acid in 15 ml ofmethanol. The obtained mixture was stirred for 15 hours at 50° C.Methanol was removed by a rotary evaporator, residue was acidified to pH1 and then solvent was removed until dry to yield5-methyl-6-oxo-1-heptanesulfonic acid. The product thus obtained (940mg, 4.5 mmol), 1.02 g (5.4 mmol) of 4-hydrazinobenzenesulfonic acid, and15 ml of acetic acid was refluxing for 16 hours. The insolubleprecipitate was filtered and the filtrate was evaporated. The obtainedresidue was column purified (RP-18, water) to yield 840 mg of the2,3-dimethyl-3-(4-sulfobutyl)-3H-5-indolesulfonic acid (IVd). δ_(H) (200MHz, DMSO-d₆): 7.66 (1H, s, arom H), 7.6 (1H, d, 8.0 Hz, arom H), 7.4(1H, d, 7.8 Hz arom H), 2.66 (2H, t, 6.8 Hz, CH ₂SO₃H), 2.32 (3H, s,2-CH ₃), 2.10-1.78 (2H, m, CH ₂), 1.53-1.26 (2H, m, CH ₂), 1.31 (3H, s,3-CH ₃), 0.83-0.41 (2H, m, CH ₂).

Synthesis of dipotassium2,3-dimethyl-1,3-di(4-sulfonatobutyl)-3H-5-indoliumsulfonate (1y)

A mixture of dipotassium2,3-dimethyl-3-(4-sulfonatobutyl)-3H-indole-5-sulfonate (1.96 g, 4.72mmol) and 6-bromohexanoic acid (2.3 g, 11.79 mmol) was heated at 135° C.for 8 hours under argon atmosphere. The raw product was columnpurificated (RP-18, water) to yield the title product as beige powder.Yield: 40% 1y. λ_(max) Ab 275 nm (water). δ_(H) (200 MHz, DMSO-d₆): 7.99(1H, s, arom H), 7.94 (1H, d, 8.2 Hz, arom H), 7.83 (1H, d, 8.2 Hz, aromH), 4.47 (2H, t, 7.1 Hz, NCH ₂), 2.87 (3H, s, 2-CH ₃), 2.38-2.15 (2H, m,CH ₂SO₃H), 2.23 (2H, t, 7.1 Hz, CH ₂COOH), 1.92-1.74 (2H, m, CH ₂),1.64-1.32 (8H, m, CH ₂), 1.53 (3H, s, 3-CH ₃), 0.86-0.42 (2H, m, CH ₂)

Other important intermediates for the synthesis of cyanine dyes aredescribed in Mujumdar et al., Bioconjugate Chem. 4(2) 105-111, 1993 andin several other patent applications (U.S. Patent Appl. US 2002/0077487A1, U.S. Pat. No. 5,569,587, U.S. Pat. No. 5,672,027, U.S. Pat. No.5,808,044 and WO 2005/044923).

EXAMPLE 3 Synthesis of 3-[2-(diethylphosphoryl)ethyl]-3H-indoleninecyanine 2

A mixture of4-[2-(-4-anilino-1,3-butadienyl)-3-(5-carboxypentyl)-3-methyl-5-sulfo-3H-1-indoliumyl]-1-butanesulfonate(270 mg, 0.42 mmol) synthesized according to Mujumdar et al.,Bioconjugate Chem. 4(2) 105-111, 1993, and indolenine derivative 1q (197mg, 0.35 mmol), 7 ml of dry pyridine and 7 ml of acetic anhydride wereheated at reflux for min. The solvent was removed under reduced pressureuntil dry. The raw product was column purified (RP-18, water) to givethe title dye as blue powder. λ_(abs) 651 nm, λ_(em) 671 nm (water).δ_(H) (200 MHz, DMSO-d₆): 8.50-8.28 (2H, m, βH), 7.82 (2H, s, arom H),7.63 (2H, d, 8.4 Hz arom H), 7.35 (2H, d, 8.4 Hz, arom H), 6.61 (1H, t,12.2 Hz, γH), 6.42 (1H, d, 13.6 Hz, αH), 6.37 (1H, d, 13.5 Hz, αH),4.19-3.96 (4H, m, NCH ₂), 3.88-3.65 (4H, m, P(CH ₂CH₃)₂), 2.59-2.42 (2H,m, CH ₂SO₃H), 2.19 (2H, t, 7.0 Hz, CH ₂COOH), 1.72 (3H, s, 3-CH₃), 1.69(6H, s, 3-CH₃), 1.80-1.60 (6H, m, CH ₂), 1.60-1.46 (4H, m, CH ₂),1.44-1.32 (2H, m, CH ₂), 1.20-1.03 (2H, m, CH ₂), 1.19-1.07 (6H, m,P(CH₂CH ₃)₂), 0.87-0.53 (2H, m, CH ₂).

EXAMPLE 4 Synthesis of 3-[2-(diethylphosphoryl)ethyl]-3H-indoleninecyanine 32-(4-anilino-1,3-butadienyl)-1-(5-carboxypentyl)-3-methyl-3-(4-sulfobutyl)-3H-5-indoliumsulfonate

A mixture of 500 mg (0.973 mmol) of indolenine 1y and 503 mg (1.946mmol) of malondialdehyde-bis-(phenylimin) monohydrochloride(Sigma-Aldrich, Cat.: 38, 353-8, Lot.: S47683-178) were refluxed withstirring for 4.5 h. After cooling to RT 2.7 mL water were added, whichwas accompanied with spontaneous heating. The solvent was removed underreduced pressure and the residue was treated with 40 mL of ethylacetate.The oily solid was filtered off, washed with ethyl acetate until thesolvent became colorless and dried over P₂O₅. The yield was almostquantitative. UV: Absorption 453 nm (water).

Dye 3 was obtained from2-(4-anilino-1,3-butadienyl)-1-(5-carboxypentyl)-3-methyl-3-(4-sulfobutyl)-3H-5-indoliumsulfonateand indolenine derivative 1q analogously to dye 2. λ_(abs)=654 nm,λ_(em)=671 nm. δ_(H) (200 MHz, DMSO-d₆): 8.50-8.29 (2H, m, 13 H), 7.80(2H, s, arom H), 7.64 (2H, d, 8.2 Hz arom H), 7.34 (2H, d, 8.2 Hz, aromH), 6.61 (1H, t, 12.2 Hz, γH), 6.40 (2H, d, 13.6 Hz, αH), 4.20-3.96 (4H,m, NCH ₂), 3.95-3.61 (4H, m, P(CH ₂CH₃)₂), 2.34-2.14 (4H, m, CH ₂SO₃H),2.19 (2H, t, 7.1 Hz, CH ₂COOH), 1.73 (3H, s, 3-CH₃), 1.66 (3H, s,3-CH₃), 1.81-1.60 (6H, m, CH ₂), 1.60-1.30 (8H, m, CH ₂), 1.26-1.05 (2H,m, CH ₂), 1.20-1.06 (6H, m, P(CH₂CH ₃)₂), 0.87-0.42 (4H, m, CH ₂).

EXAMPLE 5

Dye 4 was obtained from potassium2-(4-anilino-1,3-butadienyl)-3-(5-carboxypentyl)-3-methyl-1-(4-sulfonatobutyl)-3H-5-indoliumsulfonateand indolenine derivative 1p analogously to the procedure described inExample 3.

Compound 4: δ_(H) (200 MHz, DMSO-d₆): 8.48-8.26 (2H, m, 3H), 7.76 (2H,s, arom H), 7.64 (2H, d, 7.7 Hz arom H), 7.38 (2H, d, 7.8 Hz, arom H),6.61 (1H, t, 12.3 Hz, γH), 6.47 (1H, d, 12.8 Hz, αH), 6.39 (1H, d, 12.1Hz, αH), 4.19-4.03 (4H, m, NCH ₂), 4.96-3.81 (4H, m, P(CH ₂CH₃)₂),2.6-2.26 (4H, m, CH ₂SO₃H), 2.04 (2H, t, 6.7 Hz, CH ₂COOH), 1.72 (3H, s,3-CH₃), 1.67 (3H, s, 3-CH₃), 1.88-1.42 (10H, m, CH ₂), 1.39-0.92 (6H, m,CH ₂), 1.12 (6H, t, 6.9 Hz, P(CH₂CH ₃)₂), 0.89-0.28 (4H, m, CH ₂).

EXAMPLE 6

Dye 5 was obtained from potassium2-(4-anilino-1,3-butadienyl)-3-(5-carboxypentyl)-3-methyl-1-(4-sulfonatobutyl)-3H-5-indoliumsulfonateand potassium2,3-dimethyl-3-(2-phosphorylethyl)-1-(4-sulfonatobutyl)-3H-5-indoliumsulfonate(1p) analogously to the procedure given for Example 3.

Compound 5: λ_(abs)=653 nm, λ_(em)=672 nm (water). δ_(H) (200 MHz,DMSO-d₆): 8.40-8.18 (2H, m, βH), 7.75 (1H, s, arom H), 7.71 (1H, s, aromH), 7.63 (2H, d, 8.5 Hz arom H), 7.33 (2H, d, 7.8 Hz, arom H), 6.61 (1H,t, 12.3 Hz, γH), 6.46 (1H, d, 13.0 Hz, αH), 6.40 (1H, d, 12.5 Hz, αH),4.19-4.03 (4H, m, NCH ₂), 2.59-2.27 (4H, m, CH ₂SO₃H), 2.05 (2H, t, 7.4Hz, CH ₂COOH), 1.72 (3H, s, 3-CH₃), 1.66 (3H, s, 3-CH₃), 1.89-1.52 (6H,m, CH ₂), 1.42-0.99 (10H, m, CH ₂), 0.95-0.35 (4H, m, CH ₂).

EXAMPLE 7

A mixture of indolenine derivative 1n (590 mg, 1.14 mmol),malondialdehyde-bis-(phenylimin) monohydrochloride (120 mg, 0.46 mmol),10 ml of dry pyridine and 10 ml of acetic anhydride were heated atreflux for min. The solvent was removed under reduced pressure untildry. The raw product was column purified (RP-18, CH₃CN—H₂O:60-80%) togive the dye as blue resin. λ_(abs) 667 nm, λ_(em) 685 nm (chloroform).δ_(H) (200 MHz, DMSO-d₆): 8.28 (2H, t, 13.1 Hz βH), 7.41 (2H, s, aromH), 7.33-7.15 (4H, m, arom H), 6.55 (1H, t, 12.2 Hz γH), 6.30 (2H, d,13.6 Hz αH), 3.98 (8H, t, 5.8 Hz POCH₂), 2.37 (3H, s, 5-CH₃), 2.27 (3H,s, 5-CH₃), 2.41-2.11 (8H, m, CH₂COOH), 1.82-0.93 (28H, m, CH₂), 1.53(6H, s, 3-CH₃), 0.81-0.42 (4H, m, CH₂).

EXAMPLE 8

The dye was obtained from indolenine derivative 1y and1-(5-Carboxypentyl)-3,3-dimethyl-2-[(E)-2-(phenylamino)ethenyl]-3H-indolium-5-sulfonate(see above) analogously to dye 7. The raw product was column purified(Silica gel 60, MeOH—CHCl₃, 15%) to give the dye as blue resin. λ_(abs)661 nm, λ_(em) 682 nm (chloroform). δ_(H) (200 MHz, DMSO-d₆): 8.50-8.27(1H, m, βH), 7.64 (2H, s, arom H), 7.42 (2H, d, 7.1 Hz, arom H), 7.28(2H, d, 7.2 Hz, arom H), 6.60 (1H, t, 12.1 Hz, γH), 6.37 (1H, d, 13.9Hz, αH), 6.33 (1H, d, 13.6 Hz, αH), 4.25-4.03 (4H, m, NCH₂), 3.97-3.73(4H, m, POCH₂), 2.20 (2H, t, 7.4 Hz, CH₂COOH), 1.73 (3H, s, 3-CH₃), 1.72(3H, s, 3-CH₃), 1.69 (6H, s, 3-CH₃), 1.81-1.31 (8H, m, CH₂), 1.25 (3H,t, 6.7 Hz, CH₃), 1.19-1.06 (6H, m, CH₃), 0.89-0.53 (2H, m, CH₂).

EXAMPLE 9

The dye was synthesized from indolenine derivative 1q and1-(5-Carboxypentyl)-3,3-dimethyl-2-[(E)-2-(phenylamino)ethenyl]-3H-indolium-5-sulfonate(see above) analogously to Example 3. The raw product was columnpurified (RP-18, CH₃CN—H₂O, 10%) to give the dye as purple powder.Yield: 5% 8. λ_(abs) 556 nm, λ_(em) 570 nm (water). δ_(H) (200 MHz,DMSO-d₆): 8.31 (1H, t, 13.0 Hz, βH), 7.81 (2H, s, arom H), 7.69 (2H, d,7.9 Hz arom H), 7.46 (1H, d, 7.1 Hz, arom H), 7.42 (1H, d, 7.3 Hz, aromH), 6.60 (2H, d, 13.3 Hz, αH), 4.23-4.04 (4H, m, NCH₂), 3.96-3.77 (4H,m, POCH₂), 2.67-2.53 (2H, m, CH₂SO₃H), 2.23 (2H, t, 6.7 Hz, CH₂COOH),1.73 (3H, s, 3-CH₃), 1.71 (6H, s, 3-CH₃), 1.88-1.64 (6H, m, CH₂),1.64-1.32 (6H, m, CH₂), 1.13 (6H, t, 7.0 Hz, CH₃), 0.98-0.68 (2H, m,CH₂).

EXAMPLE 10 Synthesis of Cyanine Dyes Having a Spacer Group in theConjugated Chain2-carboxymethylsulfanyl-3-phenylimino-1-propenyl(phenyl)ammonium bromide

A mixture of 560 mg (1.85 mmol) of 2-mercaptoacetic acid and 345 mg (4.1mmol) of NaHCO₃ in 3 mL water was heated with stirring at 60° C. for 1h. Then a solution of 573 mg (1.5 mmol) of2-bromo-3-phenylimino-1-propenyl(phenyl)ammonium bromide (synthesized asdescribed in EP1221465A1) in 20 mL of methanol was added at 60° C., themixture was stirred at this temperature for 5 h, cooled to RT, pouredinto a mixture of 50 g of ice and 1 mL of concentrated hydrochloricacid. The obtained yellow precipitate was filtered and dried.

Dye 9

A mixture of 113 mg (0.2 mmol) of indolenine 1q and 39 mg (0.1 mmol) of2-carboxymethylsulfanyl-3-phenylimino-1-propenyl(phenyl)ammonium bromidewas refluxed with stirring for 4 h in a mixture of 3 mL acetic anhydrideand 3 ml pyridine. The solvent was evaporated under a reduced pressureand the product was column purified (Silica gel RP-18). Compound 9:λ_(abs)=645 nm, λ_(em)=666 nm (water).

EXAMPLE 11 Synthesis of the symmetrical heptamethin-cyanine 10

Glutaconaldehyde bis(phenylimine) hydrochloride (143 mg, 0.5 mmol) isdissolved in a hot mixture of acetic anhydride (4 mL) and 1 mL ofpyridine and 1 mmol of 1r is added and the mixture is heated for anadditional minutes and then cooled. The solvent was removed underreduced pressure until dry. Raw product was column purified (RP-18,water) to give the title compound 10.

EXAMPLE 12 Synthesis of the sulfo-phenoxy compounds (12) Synthesis ofchloro-dye precusor (11)

A mixture of 5 mmols of 1q, and 1y,N-[(3-anilinoethylene)-2-chloro-1-cyclohexene-1-yl)-methylene]anilinemonohydro-chloride (1.30 g, 5 mmol) and sodium acetate (1.1 g, 13 mmol)is refluxed in 30 mL of dry ethanol for 1 h. Subsequently the reactionis cooled to RT and the solvent is removed under reduced pressure andthe residue is purified by reversed phase C-18 column chromatographyusing methanol-water mixtures as eluent.

Synthesis of the sulfo-phenoxy compound (12)

A cooled suspension (0° C.) of 60% of sodium hydride (120 mg, 3 mmol of100% NaH) in 10 mL of dry DMF is added to a cooled DMF solution of4-hydroxybenzene sulfonic acid dihydrate (2 mmole) under nitrogenatmosphere. After about min the reaction temperature is increased to RTand after 20 min at RT the mixture is transferred into a flaskcontaining the chloro-dye 11 (1 mmol) in 30 mL of DMF and the solutionis vigorously stirred at RT for about min to 1 h. The reaction mixtureis quenched with dry ice and the DMF was removed under reduced pressure.The crude product was purified by reversed phase C-18 columnchromatography using water as eluent.

EXAMPLE 13 Synthesis of NHS-esters

a) With TSTU (N,N,N′,N′-Tetramethyl(Succinimido)Uronium TetrafluoroBorate)

26 μl (0.15 mmol) of diisopropylethylamine and 38 mg (0.6 mmol) of TSTUare added to a mixture of 0.05 mmol 3 in 1 mL of DMF, 1 mL of dioxane,and 0.5 mL of water. After min, the mixture is filtered, and thesolvents are removed in vacuum. The product is dried over P₂O₅ and usedwithout further purification.

b) With NHS/DCC

1 mL of anhydrous DMF is added to a mixture of 0.023 mmol of 4, 14 mg(0.069 mmol) of dicyclohexylcarbodiimide (DCC), and 8 mg (0.069 mmol) ofN-hydroxysuccinimide (NHS). The solution is stirred for 24 h at roomtemperature and then filtered. The solvent is removed in vacuum, and theproduct is triturated with ether and dried over P₂O₅.

EXAMPLE 14 General Protein Labelling Procedures and Determination ofDye-to-Protein Ratios

Protein labelling reactions are carried out using a 50 mM bicarbonatebuffer (pH 9.1). A stock solution of 1 mg of dye in 100 μL of anhydrousDMF is prepared. 10 mg of protein is dissolved in 1 mL of 100 mMbicarbonate buffer (pH 9.1). Dye from the stock solution is added, andthe mixture is stirred for 8-12 h at room temperature.

Unconjugated dye is separated from labeled protein using gel permeationchromatography with SEPHADEX G50 (0.5 cm×20 cm column) and a 22 mMphosphate buffer solution (pH 7.3) as the eluent. The first colored bandcontains the dye-protein conjugate. The blue band with the much higherretention time contains the separated free dye. A series of labelingreactions using different dye-to-protein starting ratios are set up toobtain different dye-to-protein ratios for the labeled protein.

The protein concentrations are determined using the BCA Protein AssayReagent Kit from Pierce (Rockford, Ill.). The dye-to-protein ratio (D/P)gives the number of dye molecules covalently bound to protein.

Covalent Attachment of 4—NHS-Ester to Polyclonal Anti-HSA

385 μL (5.2 mg/mL) of anti-HSA is dissolved in a 750 μL bicarbonatebuffer (0.1 M, pH 9.0). 1 mg of NHS-ester of dye 4 is dissolved in 50 μLof DMF and slowly added to the above-prepared protein solution withstirring. After 20 h of stirring, the protein-conjugate is separatedfrom the free dye using Sephadex G50 and a phosphate buffer (22 mM, pH7.2). The first blue band that is isolated contains the labeledconjugate.

Conjugation of 3—NHS to HSA

0.5 mg of the NHS ester of dye 3 in 50 μL of DMF are slowly added to astirred solution of 5 mg of HSA in 750 μL of bicarbonate buffer (0.1 M,pH 9.0). The mixture is stirred for another 6 h at room temperature. Themixture is dialyzed against a phosphate buffer (22 mM, pH 7.2) using adialysis membrane (1500 FT, Union Carbide) with a cutoff of 10.000.

The labeling procedures of alternative reporter compounds havingreactive functional groups are analogous to the ones reported here.

EXAMPLE 15

Photostability of Representative Dyes of this Invention

The relative photostability of selected dyes is measured as a decreaseof their long-wavelength absorption maximum upon exposure to light. Thephotostability is compared to Cy5, a long-wavelength standard and aSulfo-dye with multiple sulfo groups, and the results are shown in FIG.1.

The relative photostability of selected dyes is measured as a decreasein fluorescence emission as upon exposure to light. The photostabilityis compared to Cy5, a long-wavelength standard and a Sulfo-dye withmultiple sulfo groups, and the results are shown in FIG. 2.

EXAMPLE 16 Selected Embodiments of the Compounds of the Invention

Description of Applications of the Invention

The reporter compounds disclosed above exhibit utility for a variety ofuseful methods for various assay formats.

The assay may be a competitive assay that includes a recognition moiety,a binding partner, and an analyte. Binding partners and analytes may beselected from the group consisting of biomolecules, drugs, and polymers,among others. In some competitive assay formats, one or more componentsare labeled with photoluminescent compounds in accordance with theinvention. For example, the binding partner may be labeled with such aphotoluminescent compound, and the displacement of the compound from animmobilized recognition moiety may be detected by the appearance offluorescence in a liquid phase of the assay. In other competitive assayformats, an immobilized enzyme may be used to form a complex with thefluorophore-conjugated substrate.

The binding of antagonists to a receptor can be assayed by a competitivebinding method in so-called ligand/receptor assays. In such assays, alabeled antagonist competes with an unlabeled ligand for the receptorbinding site. One of the binding partners can be, but not necessarilyhas to be, immobilized. Such assays may also be performed inmicroplates. Immobilization can be achieved via covalent attachment tothe well wall or to the surface of beads.

Other preferred assay formats are immunological assays. There areseveral such assay formats, including competitive binding assays, inwhich labeled and unlabeled antigens compete for the binding sites onthe surface of an antibody (binding material). Typically, there areincubation times required to provide sufficient time for equilibration.Such assays can be performed in a heterogeneous or homogeneous fashion.

Sandwich assays may use secondary antibodies and excess binding materialmay be removed from the analyte by a washing step.

Other types of reactions include binding between avidin and biotin,protein A and immunoglobulins, lectins and sugars (e.g., concanavalin Aand glucose).

Certain dyes of the invention are charged due to the presence sulfonicgroups. These compounds are impermeant to membranes of biological cells.In these cases treatments such as electroporation and shock osmosis canbe used to introduce the dye into the cell. Alternatively, such dyes canbe physically inserted into the cells by pressure microinjection, scrapeloading etc.

The reporter compounds described here also may be used to sequencenucleic acids and peptides. For example, fluorescently-labeledoligonucleotides may be used to trace DNA fragments. Other applicationsof labeled DNA primers include fluorescence in-situ hybridizationmethods (FISH) and for single nucleotide polymorphism (SNIPS)applications, among others.

Multicolor labeling experiments may permit different biochemicalparameters to be monitored simultaneously. For this purpose, two or morereporter compounds are introduced into the biological system to reporton different biochemical functions. The technique can be applied tofluorescence in-situ hybridization (FISH), DNA sequencing, fluorescencemicroscopy, and flow cytometry. One way to achieve multicolor analysisis to label biomolecules such as nucleotides, proteins or DNA primerswith different luminescent reporters having distinct luminescenceproperties. Luminophores with narrow emission bandwidths are preferredfor multicolor labeling, because they have only a small overlap withother dyes and hence increase the number of dyes possible in amulticolor experiment. Importantly, the emission maxima have to be wellseparated from each other to allow sufficient resolution of the signal.A suitable multicolor triplet of fluorophores would include a Cy3-analogof this invention, TRITC, and a Cy5-analog as described herein, amongothers.

Phosphoramidites are useful functionalities for the covalent attachmentof dyes to oligos in automated oligonucleotide synthesizers. They areeasily obtained by reacting the hydroxyalkyl-modified dyes of theinvention with 2-cyanoethyl-tetraisopropyl-phosphorodiamidite and 1-Htetrazole in methylene chloride.

The simultaneous use of FISH (fluorescence in-situ hybridization) probesin combination with different fluorophores is useful for the detectionof chromosomal translocations, for gene mapping on chromosomes, and fortumor diagnosis, to name only a few applications. One way to achievesimultaneous detection of multiple sequences is to use combinatoriallabeling. The second way is to label each nucleic acid probe with aluminophore with distinct spectral properties. Similar conjugates can besynthesized from this invention and can be used in a multicolormultisequence analysis approach.

In another approach the dyes of the invention might be used to directlystain or label a sample so that the sample can be identified and orquantitated. Such dyes might be added/labeled to a target analyte as atracer. Such tracers could be used e.g. in photodynamic therapy wherethe labeled compound is irradiated with a light source and thusproducing singlet oxygen that helps to destroy tumor cells and diseasedtissue samples.

The reporter compounds of the invention can also be used for screeningassays for a combinatorial library of compounds. The compounds can bescreened for a number of characteristics, including their specificityand avidity for a particular recognition moiety.

Assays for screening a library of compounds are well known. A screeningassay is used to determine compounds that bind to a target molecule, andthereby create a signal change which is generated by a labeled ligandbound to the target molecule. Such assays allow screening of compoundsthat act as agonists or antagonists of a receptor, or that disrupt aprotein-protein interaction. It also can be used to detect hybridizationpr binding of DNA and/or RNA.

Other screening assays are based on compounds that affect the enzymeactivity. For such purposes, quenched enzyme substrates of the inventioncould be used to trace the interaction with the substrate. In thisapproach, the cleavage of the fluorescent substrate leads to a change inthe spectral properties such as the excitation and emission maxima,intensity and/or lifetime, which allows to distinguish between the freeand the bound luminophore.

The reporter compounds disclosed above may also be relevant to singlemolecule fluorescence microscopy (SMFM) where detection of single probemolecules depends on the availability of a fluorophore with highfluorescence yield, high photostability, and long excitation wavelength.

The dye compounds are also useful for use as biological stains. The dyesare not harmful and non-toxic to cells and other biological components.There may be limitations in some instances to the use of the abovecompounds as labels. For example, typically only a limited number ofdyes may be attached to a biomolecules without altering the fluorescenceproperties of the dyes (e.g. quantum yields, lifetime, emissioncharacteristics, etc.) and/or the biological activity of thebioconjugate. Typically quantum yields may be reduced at higher degreesof labeling. Encapsulation into beads offers a means to overcome theabove limitation for the use of such compounds as fluorescent markers.Fluorescent beads and polymeric materials are becoming increasinglyattractive as labels and materials for bioanalytical and sensingapplications. Various companies offer particles with defined sizesranging from nanometers to micrometers. Noncovalent encapsulation inbeads may be achieved by swelling the polymer in an organic solvent,such as toluene or chloroform, containing the dye. Covalentencapsulation may be achieved using appropriate reactive functionalgroups on both the polymer and the dyes.

Compounds claimed here may be also used for covalent and non-covalentstains of proteins and other biomolecules in gel-electrophoresisapplications.

In general, hydrophobic versions of the invention may be used fornon-covalent encapsulation in polymers, and one or more dyes could beintroduced at the same time. Surface-reactive fluorescent particlesallow covalent attachment to molecules of biological interest, such asantigens, antibodies, receptors etc. Hydrophobic versions of theinvention such as dye having lipophilic substituents such asphospholipids will non-covalently associate with lipids, liposomes,lipoproteins. They are also useful for probing membrane structure andmembrane potentials.

Compounds of this invention may also be attached to the surface ofmetallic nanoparticles such as gold or silver nanoparticles. It hasrecently been demonstrated that fluorescent molecules may show increasedquantum yields near metallic nanostructures e.g. gold or silvernanoparticles (O. Kulakovich et al., Nanoletters 2 (12) 1449-52, 2002).This enhanced fluorescence may be attributable to the presence of alocally enhanced electromagnetic field around metal nanostructures. Thechanges in the photophysical properties of a fluorophore in the vicinityof the metal surface may be used to develop novel assays and sensors. Inone example the nanoparticle may be labeled with one member of aspecific binding pair (antibody, protein, receptor etc) and thecomplementary member (antigen, ligand) may be labeled with a fluorescentmolecule in such a way that the interaction of both binding partnersleads to an detectable change in one or more fluorescence properties(such as intensity, quantum yield, lifetime, among others). Replacementof the labeled binding partner from the metal surface may lead to achange in fluorescence that can then be used to detect and/or quantifyan analyte.

Gold colloids can be synthesized by citrate reduction of a dilutedaqueous HAuCl₄ solution. These gold nanoparticles are negatively chargeddue to chemisorption of citrate ions. Surface functionalization may beachieved by reacting the nanoparticles with thiolated linker groupscontaining amino or carboxy functions. In another approach, thiolatedbiomolecules are used directly for coupling to these particles.

In a study researchers made the observation that the fluorescencesignals of cyanine dyes such as CY5 dye and the ALEXA 647 dyes inmicroarrays are strongly dependent on the concentration of ozone duringposthybridization array washing (T. Fare et al., Anal. Chem. 75(17),4672-4675, 2003). Controlled exposures of microarrays to ozone confirmedthis factor as the root cause, and showed the susceptibility of a classof cyanine dyes (e.g., CY5 dyes, ALEXA 647 dyes) to ozone levels as lowas 5 ppb for periods as short as 10 s.

One of the significant findings was the low dose level (ozoneconcentration multiplied by exposure time) that could induce the onsetof the phenomenon, suggesting many labs may be at risk. For example, itis not uncommon that the environmental ozone levels would exceed 60 ppbduring peak traffic hours on a sunny summer afternoon. Reportercompounds present on or in arrays that are exposed to these levels foras short as 1 min may begin to show significant degradation in a typicallaboratory setting.

There are ways that help to eliminate the occurrence of ozone effects onmicroarrays, for example equipping laboratories with HVAC systems havingfilters to significantly reduce ozone levels, or the use ofdye-protecting solutions to avoid signal degradation. However, each ofthese approaches may add additional costs and/or time to perform theassay. These findings suggest the need for dyes and labels in the 600 to700 nm wavelength range with improved chemical and photochemicalstability.

Experimental data on cyanine dyes indicate that introduction ofelectron-withdrawing groups into the dye backbone may increase thephotostability of such dyes.

Analytes

The invention may be used to detect an analyte that interacts with arecognition moiety in a detectable manner. As such, the invention can beattached to a recognition moiety which is known to those of skill in theart. Such recognition moieties allow the detection of specific analytes.Examples are pH-, or potassium sensing molecules, e.g., synthesized byintroduction of potassium chelators such as crown-ethers (aza crowns,thia crowns etc). Dyes with N—H substitution in the heterocyclic ringsare known to exhibit pH-sensitive absorption and emission (S. Miltsov etal., Tetrahedron Lett. 40: 4067-68, (1999), M. E. Cooper et al., J.Chem. Soc. Chem. Commun. 2000, 2323-2324), Calcium-sensors based on theBAPTA (1,2-Bis(2-aminophenoxy)ethan-N,N,N′,N′-tetra-aceticacic)chelating moiety are frequently used to trace intracellular ionconcentrations. The combination of a compound of the invention and thecalcium-binding moiety BAPTA may lead to new long-wavelength absorbingand emitting Ca-sensors which could be used for determination of intra-and extracellular calcium concentrations (Akkaya et al. TetrahedronLett. 38:4513-4516 (1997). Additionally, or in the alternative, reportercompounds already having a plurality of carboxyl functional groups maybe directly used for sensing and/or quantifying physiologically andenvironmentally relevant ions.

Fluorescence Methods

The disclosed reporter compounds may be detected using commonintensity-based fluorescence methods. The squaraine dyes are known tohave lifetimes in the range of hundreds of ps to a few ns (see Example16). The nanosecond lifetime and long-wavelength absorption and emissionof these dyes when bound to proteins may allow them to be measured usingrelatively inexpensive instrumentation that employs laser diodes forexcitation and avalanche photodiodes for detection. Typical assays basedon the measurement of the fluorescence lifetime as a parameter includefor example FRET (fluorescence resonance energy transfer) assays. Thebinding between a fluorescent donor labeled species (typically anantigen) and a fluorescent acceptor labeled species may be accompaniedby a change in the intensity and the fluorescence lifetime. The lifetimecan be measured using intensity- or phase-modulation-based methods (J.R. LAKOWICZ, PRINCIPLES OF FLUORESCENCE SPECTROSCOPY (2^(nd) Ed. 1999)).

Cyanine dyes exhibit high intrinsic polarization in the absence ofrotational motion, making them useful as tracers in fluorescencepolarization (FP) assays. Fluorescence polarization immunoassays (FPI)are widely applied to quantify low molecular weight antigens. The assaysare based on polarization measurements of antigens labeled withfluorescent probes. The requirement for polarization probes used in FPIsis that emission from the unbound labeled antigen be depolarized andincrease upon binding to the antibody. Low molecular weight specieslabeled with the compounds of the invention can be used in such bindingassays, and the unknown analyte concentration is determined by thechange in polarized emission from the fluorescent tracer molecule.

Compositions and Kits

The invention also provides compositions, kits and integrated systemsfor practicing the various aspects and embodiments of the invention,including producing the novel compounds and practicing of assays. Suchkits and systems may include a reporter compound as described above, andmay optionally include one or more of solvents, buffers, calibrationstandards, enzymes, enzyme substrates, and additional reporter compoundshaving similar or distinctly different optical properties.

Although the invention has been disclosed in preferred forms, thespecific embodiments thereof as disclosed and illustrated herein are notto be considered in a limiting sense, because numerous variations arepossible. Applicant regards the subject matter of his invention toinclude all novel and nonobvious combinations and subcombinations of thevarious elements, features, functions, and/or properties disclosedherein. No single element, feature, function, or property of thedisclosed embodiments is essential. The following claims define certaincombinations and subcombinations of elements, features, functions,and/or properties that are regarded as novel and nonobvious. Othercombinations and subcombinations may be claimed through amendment of thepresent claims or presentation of new claims in this or a relatedapplication. Such claims, whether they are broader, narrower, or equalin scope to the original claims, also are regarded as included withinthe subject matter of applicant's invention.

We claim:
 1. A composition comprising a reporter compound having theformula:

and its salts, or

and its salts, or

and its salts, where: n is an integer from 0 to 3; each R¹ independentlyis —H, a linked reactive group, a linked ionic group, a linkedconjugated substance, a linked phosphonate group, a linked substitutedalkyl phosphonate group, or an aliphatic group optionally substituted by1-6 heteroatoms independently selected from the group of N, O, and S; Zis

each R^(τ)independently is -H, a linked reactive group, a linked ionicgroup, a linked conjugated substance, or an aliphatic group optionallysubstituted by 1-6 heteroatoms independently selected from the group ofN, O, and S; each R³ independently is —H, a sulfo group, or an aliphaticgroup optionally substituted by 1-6 heteroatoms independently selectedfrom the group of N, O, and S; each R^(A) independently is —H, or analiphatic group optionally substituted by 1-6 heteroatoms independentlyselected from the group of N, O, and S; each R^(P) independently is —H,a linked phosphonate group, a linked substituted alkyl phosphonategroup, a linked reactive group, a linked ionic group, a linkedconjugated substance, or an aliphatic group optionally substituted by1-6 heteroatoms independently selected from the group of N, O, and S;wherein at least one R^(P) is a linked substituted alkyl phosphonategroup or a linked phosphonate group except when at least one R¹ is alinked substituted alkyl phosphonate group or a linked phosphonategroup; provided that one R^(P) is a linked reactive group or a linkedconjugated substance S_(c), when the other R^(P) or at least one R¹ is alinked phosphonate group; provided that at least one of R¹ and R³contains a sulfo group except when at least one R^(P) substituent is alinked substituted alkyl phosphonate group; wherein each linkedphosphonate group, when present, independently is -L-PO₃ ^(⊖), -L-PO₃^(2⊖), or -L-PO(OH)₂; wherein each linked substituted alkyl phosphonate,when present, independently is -L-PO₂(OR^(M))^(⊖), or -L-PO(OR^(M))₂,where each R^(M) independently is H, a linked reactive group, a linkedionic group, or a linked conjugated substance, provided that at leastone R^(M) is a linked reactive group, a linked ionic group, or a linkedconjugated substance; and wherein L is an aliphatic group optionallysubstituted by 1-6 heteroatoms independently selected from the group ofN, O, and S.
 2. The composition of claim 1, wherein at least one R^(P)is —(CH₂)_(m) PO₃(R^(N))^(⊖), or —(CH₂)_(m)PO₃(R^(N))₂; wherein m is aninteger from 1 to 10; and wherein each R^(N) independently is H, alinked reactive group, a linked ionic group, or a linked conjugatedsubstance.
 3. The composition of claim 1, wherein at least one of R¹,R^(τ), and R^(P) comprises a reactive group independently selected fromthe group consisting of an acylamide, an activated ester of a carboxylicacid, an acyl nitrile, an aldehyde, an alkyl halide, an alkyne, ananhydride, an aniline, an aryl halide, an aziridine, an azide, aboronate, a carboxylic acid, a diazoalkane, a cycloolefin, ahaloacetamide, a halotriazine, a hydrazone, an imido ester, anisothiocyanate, an isocyanate, a maleimide, a phosphoramidite, apyrylium moiety, a reactive platinum complex, a sulfuryl halide, a thiolgroup, and a photoactivatable group.
 4. The composition of claim 1,wherein at least one of R¹, R^(τ), and R^(P) comprises an reactive groupindependently selected from the group consisting of anN-hydroxysuccinimide ester, an isothiocyanate, a sulfonylhalogenide, anazide, an iodoacetamide and a maleimide.
 5. The composition of claim 1,wherein at least one of R¹, R^(τ), and R^(P) comprises a conjugatedsubstance.
 6. The composition of claim 5, wherein at least one of R¹,R^(τ), and R^(P) comprises a conjugated substance independently selectedfrom the group consisting of an antibody, a protein, a phycobiliprotein,a polypeptide and a peptide.
 7. The composition of claim 5, wherein atleast one of R¹, R^(τ), and R^(P) comprises a conjugated substanceindependently selected from the group consisting of a nucleotide, apolynucleotide, a bead, a microplate well surface, a phospholipid, ametallic nanoparticle, an amino acid, a nucleic acid, a protein nucleicacid, a sugar, a polysaccharide, an oligosaccharide, a fluorescent dye,a non-fluorescent dye, and a reporter compound.
 8. The composition ofclaim 1, wherein at least one of R¹, R³, R^(τ), and R^(P) comprises anionic group, wherein the ionic group increases the hydrophilicity of theentire compound.
 9. The composition of claim 1, wherein at least one ofR¹, R³, R^(τ), and R^(P) comprises an ionic group independently selectedfrom the group consisting of —SO₃ ^(⊖), —OSO₃ ^(⊖), —COO^(⊖), —PO₃^(2⊖), —OPO₃ ^(2⊖), —PO₃(R₅)^(⊖), —OPO₃(R₅)^(⊖), and —N(R⁵)₃ ^(⊕);wherein each R⁵ independently is —H, a linked reactive group, a linkedconjugated substance, an aromatic group, or an aliphatic groupoptionally substituted by 1-6 heteroatoms independently selected fromthe group of N, O, and S.
 10. The composition of claim 1, wherein thereporter compound is a first reporter compound, and further comprising asecond reporter compound selected from the group consisting of aluminophore and a chromophore.
 11. The composition of claim 10, whereinone of the first reporter compound and the second reporter compound is aFörster resonance energy transfer (FRET) donor and the other of thefirst reporter compound and the second reporter compound is a FRETacceptor.
 12. The composition of claim 10, wherein the second reportercompound is a phycobiliprotein.
 13. The composition of claim 1, whereinthe linked reactive group, the linked ionic group, the linked conjugatedsubstance, the linked phosphonate group, and the linked substitutedalkyl phosphonate group, when present, are each linked by an independentlinking group, wherein each independent linking group independently isan aliphatic group optionally substituted by 1-6 heteroatomsindependently selected from the group of N, O, and S, or a polyether(CH₂ 13 CH₂—O)_(m), or (CH₂)_(m)—NH—CO—(CH₂)_(m), wherein m is aninteger from 1 to
 10. 14. The composition of claim 1, wherein thereporter compound has the formula:

and its salts, or

and its salts, or

where: each R³ independently is H, alkyl, or sulfo; R^(P) is alkyl, alinked reactive group, or a linked conjugated substance S_(c); each R⁷independently is H, a linked reactive group, a linked ionic group, or alinked conjugated substance; each X independently is —H, a linkedreactive group, a linked ionic group, a linked conjugated substanceS_(c), a linked phosphonate, a linked substituted alkyl phosphonategroup, or SO₃H, provided that at least one X is SO₃H when both R⁷ are H;S_(c) is an antibody, an antibody fragment, a protein, a fluorescentprotein, a lectin, a nucleotide, an oligonucleotide, a peptide, apolypeptide, a nanoparticle, a protein nucleic acid, a small drug, aphospholipid, a metallic semiconductor, a metallic dielectricnanoparticle, a nanotube, an amino acid, a nucleic acid, a sugar, apolysaccaride, an oligosaccharide, a second fluorescent ornon-fluorescent dye, or a tyramide; each s independently is an integerfrom 1 to 6; each t independently is an integer from 0 to 6; n is aninteger from 0 to 3; and provided that R^(P) is a linked reactive groupor a linked conjugated substance S_(c) when both R⁷ are H.
 15. Thecomposition of claim 1, wherein the reporter compound has the formula:

or its diastereomers, where R is independently a reactive group or alinked conjugated substance S_(c); R⁷ is independently H, a linkedreactive group, a linked conjugated substance S_(c), or a linked ionicgroup; R³ is independently H, alkyl, or SO₃H; n is 0 to 3; r is 1 to 10;s is 1 to 10; t is independently 1 to 4; and X is independently H orSO₃H, provided that at least one X is SO₃H when both R₇ are H.
 16. Thecomposition of claim 1, wherein the reporter compound has the formula:

or its diastereomers, where R is independently a reactive group or alinked conjugated substance S_(c); R⁷ is independently H, a linkedreactive group, a linked conjugated substance S_(c), or a linked ionicgroup; R³ is independently H, alkyl, or SO₃H; n is 0 to 3; r is 1 to 10;s is 1 to 10; t is independently 1 to 4; and X is independently H orSO₃H, provided that at least one X is SO₃H when both R⁷ are H.
 17. Thecomposition of claim 1, wherein the reporter compound has the formula:

R³ is independently H, alkyl, or SO₃H; R⁷ is independently H, a linkedreactive group, a linked conjugated substance S_(c), or a linked ionicgroup; R⁸ is independently H, a linked reactive group, a linkedconjugated substance S_(c), or a linked ionic group; where R⁹ isindependently H, alkyl, a linked reactive group, or a linked conjugatedsubstance S_(c), provided that R⁹ is a linked reactive group or a linkedconjugated substance S_(c) when R⁷ and R⁸ are H; n is 0 to 3; t isindependently 1 to 10; and X is independently H, SO₃H, a linkedconjugated substance Sc, a reactive group, or a linked substituted alkylphosphonate group, provided that at least one of X and R₃ is SO₃H whenR⁷ and R⁸ are H.
 18. The composition of claim 1, wherein the reportercompound has the formula:

R³ is independently H, alkyl or SO₃H; R⁷ is independently H, a linkedreactive group, a linked conjugated substance S_(c), or a linked ionicgroup; R⁸ is independently H, a linked reactive group, a linkedconjugated substance S_(c), or a linked ionic group; where R⁹ is H,alkyl, a linked reactive group, or a linked conjugated substance S_(c),provided that R⁹ is a linked reactive group or a linked conjugatedsubstance S_(c) when R⁷ and R⁸ are H; n is 0 to 3; t is independently 1to 10; and X is independently H, SO₃H, a linked conjugated substance Sc,a reactive group, or a linked substituted alkyl phosphonate group,provided that one of X and R₃ is SO₃H when R⁷ and R⁸ are H.
 19. Thecomposition of claim 1, wherein the reporter compound has the formula:

R³ is independently H, alkyl, or SO₃H; R⁷ is independently H, a linkedreactive group, a linked conjugated substance S_(c), or a linked ionicgroup; R⁸ is independently H, a linked reactive group, a linkedconjugated substance S_(c), or a linked ionic group; n is 0 to 3; t isindependently 1 to 10; and R^(P) is an alkyl group, a linked reactivegroup, a linked ionic group, or a linked conjugated substance S_(c); andR is a linked reactive group or a linked conjugated substance S_(c).